Mesenchymal Stem Cells Conditioned Medium and Methods of Generating and Using the Same

ABSTRACT

Mesenchymal stem cell conditioned medium and methods for preparing the conditioned medium are provided. The methods comprise culturing MSCs in a medium comprising nicotinamide or nicotinamide and fibroblast growth factor 4 (FGF4) and collecting the conditioned medium. Compositions comprising the mesenchymal stem cell conditioned medium and uses thereof are also provided.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to theproduction and use of culture medium from mesenchymal stem cell culture.

Mesenchymal stem cells (MSCs) are non-hematopoietic cells that arecapable of differentiating into specific types of mesenchymal orconnective tissues including adipose, osseous, cartilaginous, elastic,neuronal, hepatic, pancreatic, muscular, and fibrous connective tissues.The specific differentiation pathway which these cells enter dependsupon various influences from mechanical influences and/or endogenousbioactive factors, such as growth factors, cytokines, and/or localmicroenvironmental conditions established by host tissues.

MSCs reside in a diverse host of tissues throughout the adult organismand possess the ability to ‘regenerate’ cell types specific for thesetissues. Examples of these tissues include adipose tissue, umbilicalcord blood, periosteum, synovial membrane, muscle, dermis, pericytes,blood, bone marrow and trabecular bone.

Even though MSCs multiply relatively easily in vitro, theirproliferative potential and their stem cell characteristics arecontinuously decreased during prolonged culture. For example, it hasbeen shown that expansion in culture leads to premature senescence (theprocess of aging characterized by continuous morphological andfunctional changes). Cells became much larger with irregular and flatshape and the cytoplasm became more granular. Thesesenescence-associated effects are continuously acquired from the onsetof in vitro culture (PLoS ONE, May 2008|Volume 3|Issue 5|e2213). As aresult, the successful manufacturing for commercialization of largebatches from one donor of homogenous MSCs that preserve theircharacteristics following expansion in culture remains a challenge.

MSC are considered immune privileged, and useful for transplantation,thus culture and expansion of large numbers of mesenchymal stem cellshas been undertaken.

When transplanted, MSCs exert their effect on other cells throughmultiple secreted bioactive factors such as cytokines, growth factorsand angiogenic factors (see Wang et al, J Hemat Oncol. 2012, 5:19).Cultured mesenchymal and mesenchymal stem cells secrete these factorsinto the culture medium, endowing the medium with potentially usefulproperties as a supplement to cell culture, as a therapeutic compositionor source thereof, and as a potentially economical adjunct oralternative to use of MSC themselves.

U.S. Pat. No. 6,642,048 to Xu et al discloses a cell free conditionedmedium from MSC cell culture for feeder-layer free culture ofpluripotent stem cells, but requires the transfection of hESCs. USPatent Application No. 2012/0251489 to Herrera Sanchez et al teaches thepreparation and use of a cell-free conditioned medium from liver stemcells for inhibition of proliferation of tumor cells. US PatentApplication No. 20110262392 to Habib et al teach the production and useof a cell free conditioned medium from a particular population ofcultured adherent bone marrow cells for modulation of apoptosis incancer cells. US Patent Application No. 20100323027 to Lim et al teachesthe production of a cell-free conditioned medium from cultures ofmesenchymal stem cells grown with added FGF 2 (FGF basic), for a varietyof therapeutic uses. Conditioned medium from cultured transgenicdifferentiating hESCs (Xu et al., 2004) and from coculture of themesenchymal stem cells with mouse OP9 cell line (Barberi et al., 2005)has been suggested, but introduced unacceptable risks of tumorigenicityor infection of xenozootic infectious agents.

Additional studies have indicated, for example, the potential ofmesenchymal stem cell conditioned medium for inhibiting lung fibrosis inpulmonary disease (Cargnoni et al, Cytotherapy 2012;14: 153-61),enhancing kidney repair in kidney disease (van Koppen et al, PLoS one2012;7:1-12), stimulating angiogenesis and fracture repair in diabeticrats (Wang et al, JTissue Eng Regen Med 2012;6:559-69), promotion ofwound healing (Yew et al Cell Transplantation, 2011;20:693-706) andreduction of infarct size in MI (Gnecchi et al, Meth Mol Biol2009;482:281-94).

Methods for increasing proliferation and survival in MSCs have beenwidely studied over the past few years and many factors have beenproposed for increasing the expansion efficiency of these cells.Different culture conditions produce cells which can condition themedium in different ways.

For example, many protocols relating to the expansion of MSCs includeculturing in the presence of basic fibroblast growth factor (b-FGF) (VetRes Commun. 2009 December; 33(8):811-21). It has been shown that b-FGFnot only maintains MSC proliferation potential, it also retainsosteogenic, adipogenic and chondrogenic differentiation potentialsthrough the early mitogenic cycles.

Vascular endothelial growth factor (VEGF) has also been shown toincrease MSC proliferation [Pons et al., Biochem Biophys Res Commun2008, 376:419-422].

Hepatocyte growth factor (HGF) has been shown to affect proliferation,migration and differentiation (Furge et al., Oncogene 2000,19:5582-5589].

Platelet derived growth factor (PDGF) shown to be a potent mitogen ofMSCs [Kang et al., J Cell Biochem 2005, 95:1135-1145].

Epidermal growth factor (EGF) and heparin-binding EGF have both beenshown to promote ex vivo expansion of MSCs without triggeringdifferentiation into any specific lineage [Tamama et al., Stem Cells2006, 24:686-695; Krampera et al., Blood 2005, 106:59-66]. In additionto its mitogenic effect on MSCs, EGF also increases the number ofcolony-forming units by 25% [Tamama et al., J Biomed Biotechnol 2010,795385].

Addition of Wnt3a by activating the canonical Wnt pathway increased bothproliferation and survival while preventing differentiation into theosteoblastic lineage in MSCs [Boland et al., J Cell Biochem 2004,93:1210-1230].

Other growth factors that can be found in conditioned medium are knownto cause mesenchymal stem cells to differentiate into specific lineages.Transforming growth factor beta (TGFβ), for example, is known toinfluence cells from the chondrogenic lineage in vivo, promoting initialstages of mesenchymal condensation, prechondrocyte proliferation,production of extracellular matrix and cartilage-specific moleculedeposition, while inhibiting terminal differentiation [Bonewald et a., JCell Biochem 1994, 55:350-357; Longobardi L, J Bone Miner Res 2006,21:626-636].

BMP-3, another member of the transforming growth factor beta family,known to enhance bone differentiation was shown to increase MSCproliferation threefold [Stewart A et al., Cell Physiol 2010,223:658-666].

Nicotinamide (NA), the amide form of niacin (vitamin B3), is abase-exchange substrate and a potent inhibitor of NAD(+)-dependentenzymes having mono- and poly-ADP-ribosyltransferase activities.ADP-ribosylation is implicated in the modification of a diverse array ofbiological processes (Corda D, Di Girolamo M. 2003;22(9):1953-1958;Rankin P W, et al., J Biol Chem. 1989;264:4312-4317; Banasik M. et al.,J Biol Chem. 1992;267:1569-1575; Ueda K, Hayaishi 0, Annu Rev Biochem.1985;54:73-100; Smith S. Trends Biochem Sci. 2001;26:174-179; Virag L,Szabo C. Pharm. Reviews. 2002;54:375-429).

WO 07/063545 discloses the use of nicotinamide for the expansion ofhematopoietic stem and/or progenitor cell populations.

WO 03/062369 discloses the use of nicotinamide, and other inhibitors ofCD38, for the inhibition of differentiation in ex-vivo expanding stemand progenitor cells. However, WO 03/062369 does not teachadministration of nicotinamide for particular time intervals or theproduction or use of conditioned medium.

U.S. Patent Application No. 20050260748 teaches isolation and expansionof mesenchymal stem cells with nicotinamide in the presence of a lowcalcium concentration.

Additional background art includes Farre et al., Growth Factors, 2007April;25(2):71-6.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of preparing a conditioned cell culturemedium, the method comprising (a) culturing a population of themesenchymal stem cells in a medium comprising nicotinamide, and (b)collecting the conditioned cell culture medium.

According to some embodiments of the present invention culturing thepopulation of mesenchymal stem cells is effected in a medium comprisingnicotinamide and fibroblast growth factor 4 (FGF4).

According to an aspect of some embodiments of the present inventionthere is provided a method of preparing a conditioned cell culturemedium, the method comprising (a) culturing a population of mesenchymalstem cells in a medium comprising nicotinamide and fibroblast growthfactor 4(FGF4) and (b) collecting the conditioned cell culture medium.

According to some embodiments of the present invention culturing iseffected in a medium comprising DMEM.

According to some embodiments of the present invention culturing iseffected in a medium comprising serum or platelet lysate.

According to some embodiments of the present invention the mesenchymalstem cells are derived from a tissue selected from the group consistingof bone marrow, adipose tissue, placenta and umbilical cord blood.

According to some embodiments of the present invention nicotinamide isselected from the group consisting of nicotinamide, a nicotinamideanalog, a nicotinamide metabolite, a nicotinamide analog metabolite andderivatives thereof.

According to some embodiments of the present invention culturing iseffected on a plastic surface and the mesenchymal stem cells areplastic-adherent cells.

According to some embodiments of the present invention the population ofMSCs is comprised of a heterogeneous population of cells.

According to some embodiments of the present invention at least 70% ofthe heterogeneous population of cells are MSCs.

According to some embodiments of the present invention a calciumconcentration of the medium is greater than 1.8 mM.

According to some embodiments of the present invention culturing iseffected for at least 1 week, or at least 3 passages.

According to some embodiments of the present invention a concentrationof the nicotinamide is 1-20 mM.

According to some embodiments of the present invention a concentrationof the FGF4 is 10-100 ng/ml.

According to some embodiments of the present invention the culturing iseffected in a medium devoid of platelet derived growth factor (PDGF) orfibroblast growth factor 2 (FGF2) or both.

According to some embodiments of the present invention culturing thepopulation of mesenchymal stem cells comprises culturing the populationof mesenchymal stem cells for a first period of time in a medium devoidof nicotinamide; and then culturing the population for a second periodof time in a medium comprising nicotinamide and FGF4.

According to some embodiments of the present invention the culturing iseffected under conditions that do not induce differentiation of themesenchymal stem cells.

According to some embodiments of the present invention the medium devoidof nicotinamide is devoid of FGF4.

According to some embodiments of the present invention the medium devoidof nicotinamide comprises FGF4.

According to some embodiments of the present invention the culturing inthe medium devoid of nicotinamide is effected for at least one day.

According to some embodiments of the present invention the culturing inthe medium devoid of nicotinamide is effected for at least one week.

According to some embodiments of the present invention the methodfurther comprises concentrating the conditioned medium.

According to some embodiments of the present invention the mesenchymalstem cells are cultured for a first period of time in a culture mediumcomprising serum followed by a second period of time in a serum-freeculture, and wherein the conditioned medium is collected from theculture of the second period of time.

According to some embodiments of the present invention the second periodof time is 24 to 48 hours.

According to an aspect of some embodiments of the present inventionthere is provided a mesenchymal stem cell conditioned medium produced bythe methods of the invention.

According to an aspect of some embodiments of the present inventionthere is provided a mesenchymal stem cell conditioned medium comprising1-20 mM nicotinamide, reduced levels of IL-6 and wherein saidconditioned medium has anti-inflammatory and mitogenic activity.

According to some embodiments of the present invention the conditionedmedium is characterized by an increased level of at least onebiologically active factor selected from the group consisting of HGF,KGF and TGFβ, compared to levels of the at least one factor inconditioned medium from mesenchymal stem cells cultured without addednicotinamide.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising theconditioned medium of the present invention, or a biologically activefraction thereof, and a pharmaceutically acceptable excipient, diluentor carrier.

According to an aspect of some embodiments of the present inventionthere is provided a cosmeceutical composition comprising the conditionedmedium of the present invention, or a biologically active fractionthereof and a cosmetically acceptable excipient, diluent or carrier.

According to some embodiments of the present invention the conditionedmedium of the present invention for use in treating an inflammatorydisease in a subject in need thereof.

According to some embodiments of the present invention the inflammatorydisease is delayed type hypersensitivity.

According to an aspect of some embodiments of the present inventionthere is provided a method of culturing cells, the method comprisingculturing the cells in a culture medium comprising the conditionedmedium of the present invention.

According to some embodiments of the present invention the cells arekeratinocytes.

According to an aspect of some embodiments of the present inventionthere is provided a cell culture comprising cells and a culture medium,the medium comprising the conditioned medium of the present invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings and images.With specific reference now to the drawings in detail, it is stressedthat the particulars shown are by way of example and for purposes ofillustrative discussion of embodiments of the invention. In this regard,the description taken with the drawings makes apparent to those skilledin the art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a bar graph illustrating that basic fibroblast growth factor(bFGF, FGF2) has a negative effect on the ability of nicotinamide toincrease proliferation of mesenchymal stem cells.

FIGS. 2A-B illustrate that heparin-binding EGF-like growth factor(HB-EGF) has a negative effect on the ability of nicotinamide toincrease proliferation of two different batches of mesenchymal stemcells.

FIGS. 3A-D are bar graphs illustrating the synergistic activity ofnicotinamide (NAM) and FGF4 on expansion of mesenchymal stem cells. Fourdifferent batches of MSC cultures were treated with FGF4 (50 ng/ml), NAM(5 mM) or a combination of FGF4+NAM. Cumulative cell counts at theindicated passages are shown.

FIG. 3E is a graph illustrating the effect of nicotinamide, with andwithout FGF4 on bone marrow-derived MSC proliferation, through 5passages of culture. Bone-marrow derived mesenchymal stem cells wereisolated using Ficoll and plastic adherence method, and cultured forseveral passages with fetal bovine serum. −NAM−FGF4=controls (light bluecircles), −NAM+FGF4=culture with 50 ng/ml FGF4 (dark blue circles), +NAM−FGF4=culture with 5 mM NAM (pink circles), +NAM+FGF4=culture with 50ng/ml FGF4 and 5 mM NAM (red circles). Note the synergic effect ofNicotinamide and FGF4 added together throughout all passages of MSCproliferation;

FIGS. 4A-B are graphs illustrating that nicotinamide (NAM) preserves theundifferentiated state of MSCs cultured with FGF4. Two different batchesof MSC cultures were treated with FGF4 (50 ng/ml), NAM (5 mM) or acombination of FGF4+NAM. Cell size was analyzed by Cedex cell counter.

FIGS. 5A-D are graphs illustrating that cells expanded with acombination of NAM+FGF4 are undifferentiated MSCs (CD105+CD45−). Fourdifferent batches of MSC cultures were treated with FGF4 (50 ng/ml), NAM(5 mM) or a combination of FGF4+NAM. Percent of MSC (CD105+CD45−) wasanalyzed by FACS.

FIGS. 6A-D are bar graphs illustrating inconsistent results obtainedfollowing expansion of MSC with NAM+PDGF-BB. Four different batches ofMSC cultures were treated with PDGF-BB (50 ng/ml), NAM (5 mM) or acombination of PDGF-BB+NAM. Cumulative cell counts at the indicatedpassages are shown.

FIGS. 7A-D are graphs illustrating that MSC cultures treated withPDGF-BB or a combination of PDGF-BB+NAM comprise a higher fraction ofcells other than MSCs that contaminates the cultures as compared to MSCcultured in the absence of PDGF-BB. Four different batches of MSCcultures were treated with PDGF-BB (50 ng/ml), NAM (5 mM) or acombination of PDGF-BB+NAM. Percent of MSC (CD105+CD45−) was analyzed byFACS.

FIGS. 8A-B are bar graphs illustrating a consistent synergistic effectbetween NAM and FGF4 in contrast to the absence of a synergistic oradditive effect between FGF4 and PDGF-BB. Further, a combination of NAM,FGF4 and PDGF-BB had an adverse effect on MSC expansion. MSC cultureswere treated with PDGF-BB (50 ng/ml), FGF4 (50 ng/ml) and NAM (5 mM) ora combination of two or three factors, as indicated. Cumulative cellcounts at the indicated passages are shown.

FIGS. 9A-B are graphs illustrating that PDGF-BB supports expansion ofcells other than MSCs in MSC cultures. This effect is not alleviated byNAM and/or FGF4. MSC cultures were treated with PDGF-BB (50 ng/ml), FGF4(50 ng/ml) and NAM (5 mM) or a combination of two or three factors, asindicated. Percent of MSC (CD105+CD45−) was analyzed by FACS.

FIGS. 10A-H are photographs of day 34 MSC cultures illustrating thatPDGF-BB supports expansion of cells other than MSC in MSC cultures. Thiseffect is not alleviated by NAM and/or FGF4. MSC cultures were treatedwith PDGF-BB (50 ng/ml), FGF4 (50 ng/ml) and NAM (5 mM) or a combinationof two or three factors, as indicated.

FIG. 11 is a bar graph illustrating % of BM derived adherent cellsexpressing mesenchymal stem cells markers in culture seeded +/−NAM,prior to the first passage. Mononuclear cells were isolated from bonemarrow using Ficoll and the “plastic adherence” method in the presenceor absence of Nicotinamide. Non-adherent cells were washed away 3-4 dayslater and the media was replaced every 3-4 days. FACS analysis wasperformed in order to obtain expression levels of surface moleculesprior to the first passage (8 days post-seeding).

FIGS. 12A-C are bar graphs illustrating phenotypic characterization ofadipose tissue derived mesenchymal stem cells after six passages indifferent concentrations of nicotinamide.

FIG. 13 is a bar graph illustrating phenotypic characterization of bonemarrow derived mesenchymal stem cells following the first passage ofcultures treated +/−different concentration of nicotinamide. Mononuclearcells were isolated from bone marrow using Ficoll and the “plasticadherence” method. Non-adherent cells were washed away 3-4 days laterand the media was replaced every 3-4 days. FACS analysis was performedin order to obtain expression levels of surface molecules following thefirst passage (8 days post-seeding).

FIG. 14 is a bar graph illustrating the effect of differentconcentrations of nicotinamide (added at passage 3, and at eachsubsequent passage) on the number of MSC at passage 6. Nicotinamidesubstantially improved adipose derived mesenchymal stem cell expansionin culture.

FIG. 15 is a graph illustrating the effect of nicotinamide, with andwithout FGF4 on adipose-derived MSC proliferation, through 4 passages ofculture. Adipose derived mesenchymal stem cells were isolated usingcollagenase digestion and plastic adherence method, and cultured forseveral passages with fetal bovine serum. −NAM−FGF4=controls (bluediamonds), +NAM−FGF4=culture with 5 mM NAM (red squares),+NAM+FGF4=culture with 50 ng/ml FGF4 and 5 mM NAM (green triangles).Note the synergic effect of Nicotinamide and FGF4 added together onadipose-derived MSC proliferation;

FIG. 16 is a graph detailing the effect of nicotinamide, with andwithout FGF4 on nucleated cell proliferation in adipose-derived MSCproliferation at passage 4. Adipose-derived mesenchymal stem cells wereisolated and cultured as described in FIG. 15. −NAM−FGF4=controls,+NAM−FGF4=culture with 5 mM NAM, +NAM+FGF4=culture with 50 ng/ml FGF4and 5 mM NAM. Note the synergic effect of nicotinamide and FGF4 togetheron proliferation of total nucleated cells in the culture;

FIG. 17 is a bar graph illustrating the beneficial effect of culturingadipose derived MSCs in the presence of nicotinamide and FGF4 on thesize of the cultured mesenchymal stem cells. Adipose-derived mesenchymalstem cells were isolated and cultured as described in FIG. 15. Cell sizewas analyzed by Cedex cell counter. −NAM−FGF4=controls,+NAM−FGF4=culture with 5 mM NAM, +NAM+FGF4=culture with 50 ng/ml FGF4and 5 mM NAM. Note the smaller size of MSC cells grown in the presenceof nicotinamide, and even smaller MSCs grown with nicotinamide and FGF4;

FIGS. 18A-C are graphs and plots illustrating the beneficial effect ofculturing in the presence of nicotinamide on cell size and granularity.FIG. 18C shows that cells grown in the presence of nicotinamide aresmaller and less granular (most of the cells are in the red circle), asoppose to cells grown without nicotinamide which are larger and moregranular (black circle). For FIG. 18A, the concentration of nicotinamideused was 5 mM.

FIGS. 19A-B are a graph and plots illustrating that mesenchymal stemcells grown in the presence of nicotinamide are less granular thanmesenchymal stem cells grown in the absence of nicotinamide underidentical conditions.

FIGS. 20A-B are photographs illustrating results of an in vitro woundhealing assay which was performed on MSCs cultured with (FIG. 20B) orwithout (FIG. 20A) nicotinamide at passage 3. Wound healing was observed4 days post wound formation.

FIG. 21 is a graph illustrating the effect of nicotinamide on bonemarrow derived mesenchymal stem cell Doubling Time. Nicotinamide wasadded from the initiation of the culture and at each subsequent passage.

FIG. 22 is a bar graph illustrating enhancement of Hepatocyte GrowthFactor (HGF) content of conditioned medium from nicotinamide andFGF4-treated MSC cultures. Bone marrow mesenchymal stem cells wereisolated using Ficoll and plastic adherence method, and cultured forseveral passages with fetal bovine serum, with added nicotinamide andFGF4 (+NAM+FGF4), with added nicotinamide (+NAM−FGF4) and without addednicotinamide or FGF (−NAM−FGF4). Twenty four hours before passage 4, themedia was changed and fresh media without fetal bovine serum or FGF4 wasadded. The cultured media from passage 4 cultures was collected andassayed for HGF content by ELISA. −NAM, −FGF4=control; +NAM−FGF4=5 mMNAM, +NAM+FGF4=5 mM NAM+50 ng/ml FGF4. Note the significant effect ofcombined FGF4 and nicotinamide on HGF secretion;

FIG. 23 is a bar graph illustrating enhancement of Transforming GrowthFactor-β (TGFβ) content of conditioned medium from nicotinamide andFGF4-treated MSC cultures. Bone marrow mesenchymal stem cells wereisolated and cultured as in FIG. 22 above. Cultured media was changed tomedium without fetal bovine serum or FGF4 24 hours prior to passage 4,collected from the passage 4 cultures and assayed for TGFβ content byELISA. −NAM, −FGF4=control; +NAM−FGF4=5 mM NAM, +NAM+FGF4=5 mM NAM+50ng/ml FGF4. Note the significant effect of combined FGF4 andnicotinamide on TGFβ secretion;

FIG. 24 is a bar graph illustrating enhancement of Keratinocyte GrowthFactor (KGF) content of conditioned medium from nicotinamide andFGF4-treated MSC cultures. Bone marrow mesenchymal stem cells wereisolated and cultured as in FIG. 22 above. Cultured media was changed tomedium without fetal bovine serum or FGF4 24 hours prior to passage 4,collected from the passage 4 cultures and assayed for KGF content byELISA. −NAM, −FGF4=control; +NAM−FGF4=5 mM NAM, +NAM+FGF4=5 mM NAM+50ng/ml FGF4. Note the significant effect of combined FGF4 andnicotinamide on KGF secretion;

FIG. 25 is a bar graph illustrating reduction of cytokine IL-6 (IL-6)content of conditioned medium from nicotinamide and FGF4-treated MSCcultures. Bone marrow mesenchymal stem cells were isolated and culturedas in FIG. 22 above. Cultured media was changed to medium without fetalbovine serum or FGF4 24 hours prior to passage 4, collected from thepassage 4 cultures and assayed for IL-6 content by ELISA. −NAM,−FGF4=control; +NAM−FGF4=5 mM NAM, +NAM+FGF4=5 mM NAM+50 ng/ml FGF4.Note the significant reduction by combined FGF4 and nicotinamide of IL-6secretion;

FIG. 26 is a bar graph illustrating the immune modulatory effect ofconditioned medium from nicotinamide-treated MSC cultures onDelayed-Type Hypersensitivity in mice. Bone marrow mesenchymal stemcells were isolated using Ficoll and plastic adherence method, andcultured for several passages with fetal bovine serum, with addednicotinamide (+NAM), and without added nicotinamide (−NAM). Twenty fourhours before passage 4, the media was replaced with fresh media withoutfetal bovine serum. Culture media from cultures with added nicotinamide(+NAM), and without added nicotinamide (−NAM), from passage 4 cultureswas collected 24 hours after medium replacement. The culture media withadded Nicotinamide (+NAM), without added nicotinamide (−NAM), or saline(Control) were applied (topically) 5 times at one hour intervals to theears of mice challenged with Oxazolone 6 days after sensitization withOxazolone. Ear thickness measured 24 hours after application indicatedenhanced inhibition of the DTH reaction with the conditioned medium fromMSC cultured with added nicotinamide (5 mM);

FIG. 27 is a bar graph illustrating the increase in immune modulatoryeffect of concentrating the conditioned medium from nicotinamide-treatedMSC cultures on Delayed-Type Hypersensitivity in mice. Bone marrowmesenchymal stem cells were isolated and cultured as in FIG. 26 above.Culture media from cultures with added nicotinamide (+NAM), and withoutadded nicotinamide (−NAM), from passage 4 cultures was collected 24hours after medium replacement, and saline (control) were concentrated10× by ultrafiltration before applying (topically) to the ears of micechallenged as described above. Ear thickness measured 24 hours afterapplication indicated further enhancement of inhibition of the DTHreaction with concentration of the conditioned medium from MSC culturedwith added nicotinamide (5 mM);

FIG. 28 is a bar graph illustrating the increase in immune modulatoryeffect of conditioned medium from nicotinamide and FGF4-treated MSCcultures on Delayed-Type Hypersensitivity in mice. Bone marrowmesenchymal stem cells were isolated as in FIG. 26 above and culturedfor several passages with fetal bovine serum, without added nicotinamideor FGF4 (−NAM−FGF4), with added FGF4 (−NAM+FGF4), and with addednicotinamide and FGF4 (+NAM+FGF4). Twenty four hours before passage 4,the media was changed and fresh media without fetal bovine serum andwithout FGF4 was added. Culture media from passage 4 cultures wascollected 24 hours after medium replacement, and stored frozen beforeapplying (topically) to the ears of mice challenged as described above.Ear thickness measured 24 hours after application indicated synergicenhancement of inhibition of the DTH reaction with the conditionedmedium from MSC cultured with added nicotinamide (5 mM) and FGF4 (50ng/ml). Control=saline, no conditioned medium. −NAM−FGF4=conditionedmedium from cultures without NAM or FGF4, −NAM+FGF4=conditioned mediumfrom cultures with 50 ng/ml FGF4, +NAM+FGF4=conditioned medium fromcultures with 50 ng/ml FGF4 and 5 mM NAM;

FIG. 29 is a bar graph illustrating the increase in immune modulatoryeffect of conditioned medium from nicotinamide and FGF4-treated MSCcultures on Delayed-Type Hypersensitivity in mice. Bone marrowmesenchymal stem cells were isolated and cultured as in FIG. 26 aboveand cultured for several passages with fetal bovine serum, with addednicotinamide (+NAM−FGF4), and with added nicotinamide and FGF4(+NAM+FGF4). Twenty four hours before passage 4, the media was changedand fresh media without fetal bovine serum and without FGF4 was added.Cultured media from passage 4 cultures, was collected, and stored as inFIG. 28 before applying (topically) to the ears of mice challenged asdescribed above. Ear thickness measured 24 hours after applicationindicated synergic enhancement of inhibition of the DTH reaction withthe conditioned medium from MSC cultured with added nicotinamide (5 mM)and FGF4 (50 ng/ml). Control=saline, no conditioned medium.+NAM−FGF4=conditioned medium from cultures with 5 mM NAM and no FGF4,+NAM+FGF4=conditioned medium from cultures with 50 ng/ml FGF4 and 5 mMNAM;

FIG. 30 is a bar graph illustrating time-dependent increase in immunemodulatory effect of conditioned medium from nicotinamide andFGF4-treated MSC cultures on Delayed-Type Hypersensitivity in mice. Bonemarrow mesenchymal stem cells were isolated and cultured as in FIG. 26above. Cultured media was changed to medium without fetal bovine serumand without FGF4 at 24 or 48 hours prior to passage 4, collected fromthe passage 4 cultures and stored frozen before applying (topically) tothe ears of mice challenged as described above. Ear thickness measured24 hours after application indicated synergic enhancement of inhibitionof the DTH reaction with the conditioned medium from MSC cultured withadded nicotinamide (5 mM) and FGF4 (50 ng/ml) (+NAM+FGF4=conditionedmedium from cultures with 50 ng/ml FGF4 and 5 mM NAM). Control=saline,no conditioned medium. +NAM+FGF4 24 hours=conditioned medium from cellscultured with nicotinamide and FGF4, collected 24 hours after mediumreplacement; +NAM+FGF4 48 hours=conditioned medium from cells culturedwith nicotinamide and FGF4, collected 48 hours after medium replacement;

FIGS. 31A-31B are bar graphs illustrating the effect of conditionedmedium from mesenchymal cells cultured with and without nicotinamide ornicotinamide and FGF4 on proliferation of keratinocytes in culture. FIG.31A: Normal human epidermal keratinocytes were cultured for one passagein growth medium, and reseeded in 90% growth medium and 10% conditionedmedium from MSC cultured with (+NAM) and without 5 mM nicotinamide(−NAM). Note the dramatic effect of on keratinocyte proliferation. FIG.31B: Normal human epidermal keratinocytes cultured for one passage ingrowth medium, and then reseeded in 90% growth medium and 10%conditioned medium from MSC cultured with (+NAM) and without 5 mMnicotinamide (−NAM) and with or without 50 ng/ml FGF4 (+FGF4). Note theincrease in proliferation with added FGF4 (+NAM+FGF4).

FIGS. 32A and 32B are bar graphs illustrating the effect of conditionedmedium from nicotinamide and FGF4-treated MSC cultures on TNFαproduction in activated MNCs. Peripheral blood MNCs were activated w/PHAand cultured with or without conditioned medium added in the indicatedproportions. TNFα was assayed by ELISA in the culture supernatant 72hours after activation. FIG. 32A is a single administration of theconditioned media—Column 1—Control-activated MNCs w/o conditionedmedium; Column 2—Control-non-activated MNCs w/o conditioned medium;Column 3—activated MNCs w/equal volume added conditioned medium−NAM−FGF4; Column 4—activated MNCs w/⅕ volume added conditioned medium−NAM−FGF4; Column 5—activated MNCs w/ 1/10 volume added conditionedmedium −NAM−FGF4; Column 6—activated MNCs w/equal volume addedconditioned medium +NAM+FGF4; Column 7—activated MNCs w/⅕ volume addedconditioned medium +NAM+FGF4; Column 8—activated MNCs w/ 1/10 equalvolume added conditioned medium +NAM+FGF4; Column 9—Controlnon-activated MNCs w/equal volume added conditioned medium −NAM−FGF4;Column 10—Control non-activated MNCs w/equal volume added conditionedmedium +NAM+FGF4. +NAM=5 mM nicotinamide. +FGF4=50 ng/ml FGF4. FIG.32B—is a 3-day-consecutive repeated administration of the conditionedmedia—Column 1—Control-activated MNCs w/o conditioned medium; Column2—Control-non-activated MNCs w/o conditioned medium; Column 3—activatedMNCs w/1.66 volume added conditioned medium −NAM−FGF4; Column4—activated MNCs w/0.83 volume added conditioned medium −NAM−FGF4;Column 5—activated MNCs w/0.28 volume added conditioned medium−NAM−FGF4; Column 6—activated MNCs w/1.66 volume added conditionedmedium +NAM+FGF4; Column 7—activated MNCs w/0.83 volume addedconditioned medium +NAM+FGF4; Column 8—activated MNCs w/0.28 equalvolume added conditioned medium +NAM+FGF4; Column 9—Controlnon-activated MNCs w/1.66 volume added conditioned medium −NAM−FGF4;Column 10—Control non-activated MNCs w/1.66 volume added conditionedmedium +NAM+FGF4. +NAM=5 mM nicotinamide. +FGF4=50 ng/ml FGF4. Note thestrong, dose dependent inhibition of the inflammatory response (TNFαrelease into the medium) by the conditioned medium from MSC grown withnicotinamide and FGF4.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates tocompositions comprising cell culture medium conditioned by culture ofmesenchymal stem cells, methods of producing the conditioned medium anduses thereof. In particular, the invention, in some embodiments, relatesto conditioned medium from mesenchymal stem cells cultured withnicotinamide or nicotinamide and fibroblast growth factor 4 (FGF4).

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

The multipotent character of mesenchymal stem cells (MSCs) make thesecells an attractive therapeutic tool and candidate for transplantation,capable of playing a role in a wide range of clinical applications inthe context of both cell and gene therapy strategies.

In addition, MSCs are attractive for clinical therapy in regenerativemedicine and inflammatory conditions due to their ability todifferentiate, provide trophic support, and modulate the innate immuneresponse. The therapeutic potential of MSC is being tested in multipleclinical trials for indications such as bone and cartilage repair,cardiac regeneration, critical limb ischemia, acute ischemic conditions,diabetes, Crohn's disease and graft vs. host disease. Efficientmesenchymal stem cell expansion protocols that do not have deleteriouseffects on the differentiation potential and target tissue engraftmentpotential of the cells are crucial to the success of any of thesestrategies.

Whilst studying the effect of growth factors on MSC expansion, thepresent inventors found that while growth factors such as basic FGF(bFGF), HB-EGF or platelet derived growth factor (PDGF) have anon-reproducible or even negative effect when cultured in the presenceof nicotinamide on mesenchymal stem cell proliferation (FIGS. 1, 2, 6),FGF4 surprisingly demonstrated a reproducible, synergistic activitytogether with nicotinamide on mesenchymal stem cellexpansion/proliferation (FIGS. 3A-3E).

In addition, the present inventors demonstrated an unexpected effect ofnicotinamide on cell size of mesenchymal stem cells cultured with FGF4,on seeding efficacy as evidenced by marker phenotype of the cells (FIGS.11-13), on expansion without induction of differentiation (FIGS. 12A-C)and on expansion of a more homogeneous, less granular population of MSCs(FIGS. 20A-C and 21A-B). Further, the present inventors showed that MSCscultured with nicotinamide proliferate more rapidly, decreasing doublingtime (see FIG. 26) and reach confluence substantially more rapidly(FIGS. 14-17, 26 and 27).

MSC cultures utilized by some embodiments of the invention preferablyinclude three groups of cells which are defined by their morphologicalfeatures: small and agranular cells (referred to as RS-1, hereinbelow),small and granular cells (referred to as RS-2, hereinbelow) and largeand moderately granular cells (referred to as mature MSCs, hereinbelow).The presence and concentration of such cells in culture can be assayedby identifying a presence or absence of various cell surface markers, byusing, for example, immunofluorescence, in situ hybridization, andactivity assays.

When MSCs are cultured under the culturing conditions of someembodiments of the invention they exhibit negative staining for thehematopoietic stem cell markers CD34, CD11B, CD43 and CD45. A smallfraction of cells (less than 10%) may be dimly positive for CD31 and/orCD38 markers. In addition, mature MSCs may be dimly positive for thehematopoietic stem cell marker, CD117 (c-Kit), moderately positive forthe osteogenic MSCs marker, Stro-1 [Simmons, P. J. & Torok-Storb, B.(1991). Blood 78, 5562] and positive for the thymocytes and peripheral Tlymphocytes marker, CD90 (Thy-1). On the other hand, the RS-1 cells arenegative for the CD117 and Strol markers and are dimly positive for theCD90 marker, and the RS-2 cells are negative for all of these markers.

Mesenchymal cells cultured with nicotinamide can secrete biologicallyactive factors into the medium. The present inventors have observed thatmedium collected from mesenchymal cells cultured with nicotinamidecomprises elevated levels of growth factors and cytokines (e.g.hepatocyte growth factor, keratinocyte growth factor, transforminggrowth factor beta) and reduced levels of pro-inflammatory factors (e.g.IL6) (see Example 8 and FIGS. 28-31). Addition of FGF4 to the mediumfurther increased the levels of growth factors in the medium, whilstfurther reducing the levels of IL6 in the culture medium (see Example8). Isolated culture medium of the present invention was observed tohave strong immune-modulating properties in-vitro (see FIGS. 32A and32B), a strong anti-inflammatory effect in-vivo (see FIGS. 26-30), aswell as enhancing proliferation of cells in culture (see FIGS. 31A-31B).

Thus, according to some aspects of some embodiments of the presentinvention, there is provided a method of preparing a conditioned culturemedium, the method comprising (a) culturing a population of mesenchymalstem cells in a medium comprising nicotinamide, and (b) collecting theconditioned cell culture medium.

According to one embodiment of this aspect of the invention, thepopulation of mesenchymal stem cells is cultured in a medium comprisingnicotinamide and FGF4.

Thus, according to other aspects of some embodiments of the presentinvention, there is provided a method of preparing a conditioned cellculture medium, comprising (a) culturing a population of mesenchymalcells in a medium comprising nicotinamide and fibroblast growth factor 4(FGF4) and (b) collecting the conditioned cell culture medium.

According to one embodiment of this aspect of the present invention, themesenchymal stem cells are human.

According to another embodiment of this aspect of the present invention,the mesenchymal stem cells are isolated from newborn humans.

Mesenchymal stem cells may be isolated from various tissues includingbut not limited to bone marrow, peripheral blood, blood, placenta (e.g.fetal side of the placenta), cord blood, umbilical cord, amniotic fluid,placenta and from adipose tissue. As used herein, the term “derivedfrom” indicates the tissue of origin of the mesenchymal stem cells.

A method of isolating mesenchymal stem cells from peripheral blood isdescribed by Kassis et al [Bone Marrow Transplant. 2006 May;37(10):967-76]. A method of isolating mesenchymal stem cells fromplacental tissue is described by Zhang et al [Chinese Medical Journal,2004, 117 (6):882-887]. Methods of isolating and culturing adiposetissue, placental and cord blood mesenchymal stem cells are described byKern et al [Stem Cells, 2006; 24:1294-1301].

Bone marrow can be isolated from the iliac crest of an individual byaspiration. Low-density BM mononuclear cells (BMMNC) may be separated bya FICOL-PAQUE density gradient or by elimination of red blood cellsusing Hetastarch (hydroxyethyl starch). Preferably, mesenchymal stemcell cultures are generated by diluting BM aspirates (usually 20 ml)with equal volumes of Hank's balanced salt solution (HBSS; GIBCOLaboratories, Grand Island, N.Y., USA) and layering the diluted cellsover about 10 ml of a Ficoll column (Ficoll-Paque; Pharmacia,Piscataway, N.J., USA). Following 30 minutes of centrifugation at2,500×g, the mononuclear cell layer is removed from the interface andsuspended in HBSS. Cells are then centrifuged at 1,500×g for 15 minutesand resuspended in a complete medium (MEM, a medium withoutdeoxyribonucleotides or ribonucleotides; GIBCO); 20% fetal calf serum(FCS) derived from a lot selected for rapid growth of MSCs (AtlantaBiologicals, Norcross, GA); 100 units/ml penicillin (GIBCO), 100 μg/mlstreptomycin (GIBCO); and 2 mM L-glutamine (GIBCO).

Adipose tissue-derived MSCs can be obtained from any fat-containingtissue, for example, from subcutaneous fat, by liposuction or syringesuction, and mononuclear cells can be isolated manually by removal ofthe fat and fat cells, or using the Celution System (CytoriTherapeutics) following the same procedure as described above forpreparation of MSCs.

As mentioned, the method comprises culturing (i.e. ex vivo or in vitro)the mesenchymal stem cells in a medium comprising nicotinamide and FGF4.

According to this aspect of the present invention, the cells arecultured under conditions that do not induce differentiation (e.g. inthe absence of differentiation factors or in the presence of anon-differentiating amount of differentiating factors).

The present invention contemplates directly culturing mesenchymal stemcells following isolation from their source or culturing populations ofcells that have been pre-selected for mesenchymal stem cells. Thus, thepresent invention contemplates culturing both heterogeneous populationsof cells which comprise the MSCs and more homogeneous populations ofcells, which have been enriched for MSCs, wherein more than 70%, morethan 80%, more than 90% or more than 95%, more than 98% thereof areMSCs. Also, contemplated is the enriching for MSCs concomitant with theculturing as further described herein below.

It will be appreciated that the composition of the heterogeneouspopulation of cells will be dependent on the source of the cells. Thus,for example, if the placenta is selected as the cell source, theheterogeneous population of cells will comprise placental cells as wellas mesenchymal stem cells. If the bone marrow is selected as the cellsource, the heterogeneous population of cells will comprise blood cells.

According to one method, the population of cells are cultured (in vitroor ex vivo) on polystyrene plastic surfaces (e.g. in a flask) so as toenrich for mesenchymal stem cells by removing non-adherent cells (i.e.non-mesenchymal stem cells). This method of enriching for MSCs may beeffected prior to the culturing in nicotinamide and FGF4, concomitantwith the culturing in nicotinamide and FGF4 and/or following theculturing in nicotinamide and FGF4. Thus, according to some aspects ofsome embodiments of the invention, the mesenchymal stem cells are“adherent” or “plastic-adherent” cells (e.g. cells remaining adhered tothe plastic surface after removal of non-adherent, non-mesenchymalcells).

Other methods of selecting for MSCs are known in the art including forexample positive selection against mesenchymal stem cell markers and/ornegative selection against hematopoietic stem and progenitor markerssuch as CD34, CD133, CD8, etc. Methods of determining proteincell-surface expression are well known in the art. Examples includeimmunological methods, such as, FACS analysis as well as biochemicalmethods (cell-surface labeling, e.g., radioactive, fluorescence,avidin-biotin).

It will be appreciated that a selecting stage may also be performedfollowing the culturing in nicotinamide and/or FGF4. This may beeffected as well as a preselection stage or instead of a preselectionstage.

As used herein, the term “growth medium” or “basal medium” refers to asolution of amino acids, vitamins, salts, and nutrients that iseffective to support the growth of cells in culture, although normallythese compounds will not support cell growth unless supplemented withadditional compounds. The nutrients include a carbon source (e.g., asugar such as glucose) that can be metabolized by the cells, as well asother compounds necessary for the cells' survival. These are compoundsthat the cells themselves cannot synthesize, due to the absence of oneor more of the gene(s) that encode the protein(s) necessary tosynthesize the compound (e.g., essential amino acids) or, with respectto compounds which the cells can synthesize, because of their particulardevelopmental state the gene(s) encoding the necessary biosyntheticproteins are not being expressed as sufficient levels. A number ofgrowth media are known in the art of mammalian cell culture, such asDulbecco's Modified Eagle Media (DMEM), Knockout-DMEM (KO-DMEM), andDMEM/F12, although any base medium that can be supplemented withnicotinamide and/or FGF4 and which supports the growth of primateprimordial stem cells in a substantially undifferentiated state can beemployed.

As used herein, the term “conditioned medium” refers to a growth mediumthat is further supplemented with soluble factors (“culture-derivedgrowth factors”) derived from mesenchymal stem cells, preferably humanmesenchymal stem cells, cultured in the medium. In some embodiments theconditioned medium is growth media conditioned by the growth of bonemarrow or adipose-derived mesenchymal stem cells, especially humanmesenchymal stem cells. Techniques for isolating conditioned medium froma cell culture are known in the art. In some embodiments of theinvention, the conditioned medium is essentially cell-free. In thiscontext, “essentially cell-free” refers to a conditioned medium thatcontains fewer than about 10%, fewer than about 5%, 1%, 0.1%, 0.01%,0.001%, and 0.0001% than the number of cells per unit volume, ascompared to the culture from which it was separated. As used herein, theterm “conditioned medium” also encompasses such a medium that has beentreated by concentration, filtration, extraction, fractionation or othermeans for preserving, increasing the potency, improving the stability,removing impurities, etc. Thus, conditioned medium includes extracts andfractions, for example, as defined below.

It will be appreciated that the present inventors have shown that thebiological activity of conditioned medium prepared according to themethods of the present invention was preserved following concentratedconditioned medium (see FIG. 27). This is significant, since theultrafiltration method employed to concentrate the conditioned mediumremoves many small molecules, such as nicotinamide. Without beinglimited to a single hypothesis, it can thus be suggested that thebiological activity of the conditioned medium is not dependent upon thepresence of the same concentrations of nicotinamide as provided in thegrowth medium. Thus, according to some embodiments of the presentinvention, the biological activity of the conditioned medium is not afunction of the concentration of nicotinamide in the conditioned medium.

Yet further, it will be appreciated that the conditioned medium can alsoinclude additional components added after isolation and collection ofthe conditioned medium, such as preservatives, anti-bacterial andantifungal agents, nutrients, biologically active agents such ascytokines and chemokines, drugs, etc. Still further, the conditionedmedium can be processed by heating, for example, pasteurization orautoclaving before use or storage. The conditioned medium can be storedas is, refrigerated or frozen. In one embodiment, the conditioned mediumis stored frozen, at about −5° to about −80° C. In another embodiment,the conditioned medium is dehydrated (e.g. desiccated, lyophilized, etc)and stored dry, and reconstituted at desired concentration (for example,with water) before use. In yet another embodiment, the dehydratedconditioned medium can be used, applied or administered in its driedform (for example, for topical application or formulation withexcipients, carriers, etc).

According to some embodiments, the conditioned medium is collectedfollowing removal of growth medium from the mesenchymal stem cellculture and replacement of fresh medium. In some embodiments, theconditioned medium is collected at least about 12 hours followingreplacement of the medium, about 24 hours, about 36 hours, about 48hours, about 60 hours or more following replacement of the medium. Thepresent inventors have shown that biological activity of the conditionedmedium is significant when collected 24 hours following replacement, andincreases with additional time in culture following replacement (seeFIG. 30). Thus, according to some embodiments, the conditioned medium iscollected 24 or 48 hours following replacement. In some embodiments themedium is replaced, and conditioned medium collected just before apassage of the cells, for example, before the 1^(st) passage, before the2^(nd) passage, before the 3^(rd) passage, before the 4^(th) passage, ormore. In one embodiment, the medium is replaced and conditioned mediumcollected before the 3^(rd) or before the 4^(th) passage.

In some embodiments, the medium used for replacement prior to collectionof conditioned medium is not identical to the growth medium used duringthe previous culture period. In some embodiments, the replacement mediumprovided prior to collection of conditioned medium (and thus theconditioned medium) is serum free medium. In another embodiment, thereplacement medium (and thus the conditioned medium) is devoid of FGF4.

Thus, according to one aspect of one embodiment of the presentinvention, the mesenchymal stem cells are cultured for a first period oftime in a culture medium comprising serum followed by culture for asecond period of time in a serum-free culture, and the conditionedmedium is collected from the culture of the second period of time. Inyet another embodiment, the cells are cultured for the first period oftime in a culture medium comprising FGF4 followed by culture for asecond period of time in culture medium (and collection of conditionedmedium therefrom) in a culture medium devoid of FGF4. In yet anotherembodiment, the culture medium of the first period comprises both serumand FGF4, and the culture medium of the second period is serum-free anddevoid of FGF4.

As used herein “nicotinamide” refers to nicotinamide as well as toproducts that are derived from nicotinamide, analogs thereof andmetabolites of nicotinamide or nicotinamide analogs, such as, forexample, NAD, NADH and NADPH.

As used herein, the phrase “nicotinamide analog” refers to any moleculethat is known to act similarly to nicotinamide. Representative examplesof nicotinamide analogs include, without limitation, benzamide,nicotinethioamide (the thiol analog of nicotinamide), nicotinic acid,α-amino-3-indolepropionic acid, and inhibitors of sirtuin family ofhistone/protein deacetylases. Examples of nicotinamide analogderivatives include, but are not limited to substituted benzamides,substituted nicotinamides and nicotinethioamides and N-substitutednicotinamides and nicotinthioamides.

In a particular embodiment, the nicotinamide is supplied at aconcentration of at least about 1 mM to 20 mM. In other embodiment, thenicotinamide concentration is supplied at a concentration of at leastabout 1 mM to 10 mM, e.g. about 2.5 mM, about 5 mM, about 7.5 mM.

Fibroblast growth factor 4, the FGF4 (map locus 11q13.3) gene product,FGF-4/HBGF-4/KFGF, is a 176 AA long protein derived by cleavage of theN-terminal 30 AAs of the precursor protein. FGF-4 contains a singleN-linked glycosylation site. Unglycosylated FGF-4 is cleaved into twoNH2-terminally truncated peptides (13 and 15 kDa) that are more activewith higher heparin affinity than wild-type protein.

According to a particular embodiment, the FGF4 is human FGF4.RecombinantFGF4 protein is commercially available (e.g. from Sigma Aldrich, whereit is produced in baculovirus and cleaved at the N-terminal to yield a148 AA protein; or from Invitrogen where it is produced in E. coli).

In a particular embodiment, the FGF4 is supplied to the culture at aconcentration of at least about 1-1000 ng/ml. In other embodiment, theFGF4 concentration is supplied at a concentration of at least about10-200 ng/ml, 10-100 ng/ml, e.g. about 50 ng/ml.

According to a particular embodiment, the culturing medium comprisingboth nicotinamide and FGF4 is devoid of additional growth factors suchas PDGF, HB-EGF or bFGF (FGF2).

It will be appreciated that when referring to a medium being devoid of aparticular component, the present invention contemplates that the mediumcomprises this component, but at a concentration which is below itsminimal activity. Thus, for example, certain media may comprise traceamounts of the above described growth factors, however, the methods ofthe present invention relate to a medium being devoid of exogenouslyadded growth factor beyond what is included in a commercial medium'sformula, or that resulting from overall adjustment of medium componentconcentrations. Thus, according to a particular embodiment, the mediumwhich comprises nicotinamide and FGF4 may comprise any one of the abovementioned additional growth factors but at a concentration less than 1ng/ml.

A typical cell medium to which the nicotinamide and FGF4 may be added isDulbecco's modified MEM (DMEM). Alternatively, the cell medium may beHam's F12. Other contemplated mediums include HEM RPMI, F-12, and thelike.

It will be noted that many of the culture media contain nicotinamide asa vitamin supplement for example, MEMα (8.19 μM nicotinamide), RPMI(8.19 μM nicotinamide), DMEM (32.78 μM nicotinamide) and Glascow'smedium (16.39 μM nicotinamide), however, the methods of the presentinvention relate to exogenously added nicotinamide supplementing anynicotinamide and/or nicotinamide moiety included the medium's formula,or that resulting from overall adjustment of medium componentconcentrations.

In an embodiment of the invention, the cell culture medium has a highcalcium concentration of more than about 1.8 mM, more than about 2 mM,or more than about 5 mM. It will be appreciated that the calciumconcentration is calculated as the total calcium concentration includingthat already present in the culture medium.

Thus, for example, if the medium is Dulbecco's modified MEM (DMEM)(which already has a calcium ion concentration of about 1.8 mM), noadditional calcium needs to be added. If the cell medium is Ham's F12which has a calcium ion concentration of about 0.9 mM, additionalcalcium should be added so the total calcium concentration is above 1.8mM. In one embodiment, the source of the additional calcium may beserum.

During the culturing, the medium can contain supplements required forcellular metabolism such as glutamine and other amino acids, vitamins,minerals and useful proteins such as transferrin, and the like. Themedium may also contain antibiotics to prevent contamination with yeast,bacteria, and fungi, such as penicillin, streptomycin, gentamicin, andthe like. If cells are to be cultured, conditions should be close tophysiological conditions (preferably, a pH of about 6 to about 8, and atemperature of about 30° C. to about 40° C.).

Normoxia or hypoxia conditions are also contemplated.

According to one embodiment, the culture medium is devoid of serum (i.e.serum free medium) and comprises serum replacements including, but notlimited to platelet lysate (during seeding and/or expansion).

According to still another embodiment the medium comprises about 10%fetal bovine serum. Human serum is also contemplated.

The present inventors have shown that conditioned medium frommesenchymal stem cell cultures, prepared according to the methods ofsome embodiments of the present invention is enriched with biologicallyactive factors and agents, as compared to conditioned medium from cellscultured without the addition of nicotinamide and/or FGF4. Inparticular, the conditioned medium of the present invention comprisesincreased levels of growth factors (HGF, KGF and TGFβ), and reducedlevels of the pro-inflammatory factor IL-6 (see Example 6 and FIGS.22-25).

Thus, according to one aspect of some embodiments of the presentinvention, there is provided a mesenchymal stem cell conditioned mediumcomprising 1-20 mM nicotinamide reduced levels of IL-6 and havinganti-inflammatory and mitogenic activity. According to some embodiments,the conditioned medium is further characterized by an increased level ofat least one biologically active factor selected from the groupconsisting of hepatocyte growth factor (HGF), keratinocyte growth factor(KGF) and transforming growth factor beta (TGFβ), compared to levels ofthe growth factors in conditioned medium from mesenchymal stem cellscultured without added nicotine or nicotine and FGF4. In someembodiments levels of the factors are about 10, about 20, about 30,about 40, about 50, about 60, about 70, about 100, about 150, about 200,about 250 percent greater than those of the growth factors inconditioned medium from mesenchymal stem cells cultured without addednicotine or nicotine and FGF4. In some embodiments, the levels of IL-6(and/or other pro-inflammatory factors) are about 10, about 20, about30, about 40, about 50, about 60, about 70, about 100, about 150, about200, about 250 or more percent less than those of the IL-6 inconditioned medium from mesenchymal stem cells cultured without addednicotine or nicotine and FGF4.

The culturing according to this aspect of the present invention may beeffected for a limited amount of time, such that no expansion takesplace (e.g. during the seeding stage only) or may be effected for longerperiods of time so as to allow for mesenchymal stem cell expansion (i.e.cell propagation), thereby obtaining increased quantities thereof.

For each round of propagation, adherent cells may be harvested usingtrypsin/EDTA or by cell scraping, and dissociated by passage through anarrow Pasteur plastic pipette, and preferably replated at a density ofabout 100 to about 10,000 cells/cm².

According to this aspect of the present invention, a period of timesufficient for cell expansion may be taken to mean the length of timerequired for at least one cell to divide.

According to one embodiment, the culturing is effected for at least oneday, at least two days, at least three days, at least four days, atleast five days, at least six days, at least one week, at least twoweeks, at least three weeks, at least four weeks or at least five weeks.

According to another embodiment, the culturing is not effected for morethan ten weeks.

According to still another embodiment, the cells are allowed to expandfor at least two population doublings, at least four populationdoublings, at least six population doublings, at least eight populationdoublings, at least ten population doublings, at least 15 populationdoublings, at least 20 population doublings, at least 25 populationdoublings, at least 30 population doublings, at least 35 populationdoublings, at least 40 population doublings, or at least 45 populationdoublings.

According to another embodiment, the cells are not allowed to expand formore than 50 population doublings.

The present invention contemplates additional methods of mesenchymalstem cell expansion as well as (or instead of) culturing in nicotinamideand FGF4.

Since the present inventors have found that when at least a portion ofthe time of the expansion process is effected in the presence ofnicotinamide, increased numbers of mesenchymal stem cells are obtained,preferably additional methods of expansion include culturing in thepresence of nicotinamide.

Thus, according to another aspect of the present invention theconditioned medium is produced by culturing a seeded population ofmesenchymal stem cells for a period of time sufficient for cellexpansion, wherein for at least a portion of the period of time theculturing is effected in a medium devoid of nicotinamide; and for atleast a second portion of the period of time, the culturing is effectedin a medium comprising nicotinamide and FGF4, and collecting theconditioned medium from the expanded cell culture.

The term “expanding” as used herein refers to increasing the number ofcells in the cell population due to cell replication.

According to this aspect of the present invention, the cells areexpanded under conditions that do not induce differentiation (e.g. inthe absence of differentiation factors).

The seeded population of undifferentiated mesenchymal stem cells may bea heterogeneous population of cells or a purified population ofmesenchymal stem cells, as further described herein above.

As mentioned, a medium being devoid of nicotinamide refers to a mediumcomprising less than the minimal effective amount of nicotinamide (e.g.less than 0.5 mM, or more preferably less than 0.05 mM). Thus mediumscomprising trace amounts of nicotinamide (as described herein above) maybe used for this aspect of the present invention. Thus, according to aparticular embodiment, the medium without exogenously added nicotinamidemay comprise, before the addition of exogenous nicotinamide as asupplement, nicotinamide at a concentration less than 0.5 mM or morepreferably less than 0.05 mM. According to one embodiment, the MSCs areat least 50% purified, at least 75% purified or at least 90% purified.

The population of mesenchymal stem cells may be seeded (and alsocultured) in any medium including those described herein above or thosedisclosed in U.S. Patent Application No. 20050260748, incorporatedherein by reference.

The time ratio of culturing in the presence of nicotinamide and FGF4:culturing in the absence of nicotinamide may vary and may include allratios from 1:99; 2:98; 3:97; 4:96, 5:95; 6:94; 7:93; 8:92; 9:91; 10:90;11:89; 12:88; 13:87; 14:86; 15:85; 16:84; 17:83; 18:82; 19:81; 20:80;21:79; 22:78; 23:77; 24:76; 25:75; 26:74 27:73; 28:72; 29:71; 30:70;31:69; 32:68; 33:67; 34:66; 35:65; 36:64; 37:63; 38:62; 39:61; 40:60;41:59; 42:58; 43:57; 44:56; 45:55; 46:54; 47:53; 48:52; 49:51; 50:50;51:49; 52:48; 53:47; 54:46; 55:45; 56:44; 57:43; 58:42; 59:41; 60:40;61:39; 62:38; 63:37; 64:36; 65:35; 66:34; 67:33; 68:32; 69:31; 70:30;71:29; 72:28; 73:27; 74:26; 75:25; 76:24; 77:23; 78:22; 79:21; 80:29;81:19; 82:18; 83:17; 84:16; 85:15; 86:14; 87:13; 88:12; 89:11; 90:10;91:9; 92:8; 93:7; 94:6; 95:5; 96:4; 97:3; 98:2; 99:1.

According to one embodiment, at least one full round of propagation iseffected in the presence of nicotinamide.

It will be appreciated that the culturing in the medium comprisingnicotinamide may be effected prior or following the culturing in themedium devoid of nicotinamide.

According to embodiments of the present invention, the medium which isdevoid of nicotinamide comprises FGF4 (either at the same or a differentconcentration as the medium which comprises nicotinamide).

According to other embodiments of the present invention, the mediumwhich is devoid of nicotinamide is further devoid of FGF4.

Further, the present inventors contemplate more than one culturing stagein the presence of nicotinamide and FGF4 interspersed with culturingstages in the absence of the nicotinamide and vice versa.

According to one embodiment, the culturing in the presence ofnicotinamide and FGF4 is effected for at least one day, at least twodays, at least three days, at least four days, at least five days, atleast six days, at least one week, at least two weeks, at least threeweeks, at least four weeks or at least five weeks.

According to another embodiment, the culturing in the absence ofnicotinamide is effected for at least one day, at least two days, atleast three days, at least four days, at least five days, at least sixdays, at least one week, at least two weeks, at least three weeks, atleast four weeks or at least five weeks.

As mentioned, mesenchymal stem cells can be selected based on theexpression of a mesenchymal stem cell surface marker. The selection orsorting may comprise selecting mesenchymal stem cells (MSC) from themixed population of cells by means of one or more of such surfacemarkers. The use of a selection or sorting step further enhances thestringency of sorting and selection specificity for MSCs and furthermorepotentially reduces possible contamination from the starting material.

Prior to sorting, the mixed cell populations are typically dispersedusing cell dispersing agents. Preferably single cell populations areobtained. Examples of agents that may be used to disperse the cellsinclude, but are not limited to collagenase, dispase, accutase, trypsin(e.g. trypsin-EDTA), papain. Alternatively, or additionally triturationmay also be performed to increase the dispersal of the cells.

According to a specific embodiment, the selecting is effected byselecting cells which express VCAM-1/CD106 (NP_(—)001069.1) above apredetermined level.

According to another embodiment, the selecting is effected by selectingcells which express at least one of CD105 (SH2), CD73 (SH3/4), CD44,CD90 (Thy-1), CD71, STRO-1, CD29, CD166, CD146, CD106 and CD271 above apredetermined level. According to a particular embodiment, the surfacemarker is stromal precursor antigen-1 (STRO-1), CD105 or VCAM (CD106).

According to still another embodiment, the selecting is effected byselecting cells which express at least one of CD34, CD11B, CD43 and CD45below a predetermined level.

A number of methods are known for selection or sorting based on antigenexpression, and any of these may be used in the selection or sortingstep described here. In particularly preferred embodiments, the analysisis achieved using a flow cytometer and the cells are subsequently sortedbased upon the specific light scattering and fluorescent characteristicsof each cell. Thus, the selection or sorting may be achieved by means offluorescence activated cell sorting (FACS). Exemplary Flow Cytometersthat may be used in this aspect of the present invention aremanufactured by companies such as Becton Dickinson (USA), BackmanCoulter (USA), Partec (Germany).

The above described cell populations are typically enriched for cellsthat do not express CD45. Thus, according to another embodiment, lessthan 10% of the mesenchymal cells express CD45 as measured by FACS.According to still another embodiment, more than 90% of the mesenchymalcells express CD90, as measured by FACS. According to still anotherembodiment, more than 95% of the mesenchymal cells express CD90, asmeasured by FACS. According to still another embodiment, more than 90%of the mesenchymal cells express CD44, as measured by FACS. According tostill another embodiment, more than 95% of the cells in the abovedescribed cell populations express CD44, as measured by FACS.

As mentioned, additional steps of culturing the mesenchymal stem cellsare contemplated by the present inventors prior to, during or followingthe protocol described herein. Such additional steps may involveculturing on a plastic surface, as described herein above and/oradditional expansion steps, for example, as described herein above reculturing in nicotinamide.

In some embodiments, the cells are selected according to cell size, forexample, by a cell counter based on Trypan Blue exclusion and graphicalanalysis. Suitable cell counters include, but are not limited to Cedexcounters (Roche Innovatis). The number of cells that may be culturedaccording to any of the methods of the present invention may be anynumber including small batches—e.g. 100×10⁴ cells to larger batches—e.g.100×10¹² or 100×10¹³ cells. When large batches are required, the cellsare typically cultured in a bioreactor (or in multi-level industrialflasks), the size of which is selected according to the number of cellsbeing cultured.

Examples of flasks and plates that may be used for growing MSCs incommercial quantities include for example Corning HYPERFlask™ CellCulture Vessel, Corning CellSTACK™ Chambers, Corning HYPERStack™ CellCulture Vessel, 40 stack chambers and NUNC Automatic Cell FactoryManipulator.

As used herein, the term “bioreactor” refers to any device in whichbiological and/or biochemical processes develop under monitored andcontrolled environmental and operating conditions, for example, pH,temperature, pressure, nutrient supply and waste removal. According toone embodiment of the invention, the basic classes of bioreactorssuitable for use with the present invention include static bioreactors,stirred flask bioreactors, rotating wall bioreactors, hollow fiberbioreactors and direct perfusion bioreactors, as further described in WO2005/007799, the contents of which are incorporated by reference.

As shown in Example 8 (see FIGS. 31A and 31B), cell culture mediumcomprising the conditioned medium of some embodiments of the presentinvention enhanced growth of cells (keratinocytes) in culture. Thus, theconditioned medium may be used alone, as a medium for cell culture, oras a supplement to other growth media for cell culture. Thus, accordingto one aspect of one embodiment of the present invention, there isprovided a cell culture comprising cells and a culture medium comprisinga mesenchymal stem cell conditioned medium prepared according to themethods of the invention. In another embodiment, there is provided acell population cultured in the cell culture. According to someembodiments, the cells may be any cell that can be grown in culture, andthe culture can be a primary culture, a cell line, etc. In oneembodiment, the cell culture is a mesenchymal stem cell culture or akeratinocyte cell culture.

The present inventors have shown that the conditioned medium of someembodiments of the present invention comprises biological activity, inparticular, anti-inflammatory and mitogenic activity (see Examples 7 and8 herein). Thus, the conditioned medium of the present invention orgenerated by the methods of the present invention may be used for avariety of purposes including research, for therapeutic uses, forcosmetic uses, for nutritional uses, for production of any of thecomponents of the condition medium and the like. Thus, according to someaspects of some embodiments of the present invention, there is provideda method of treating a disease or disorder in a subject in need thereof,the method comprising administering to a subject in need thereof atherapeutically effective amount of the conditioned medium the presentinvention. It will be noted that mesenchymal stem cell conditionedmedium, comprising any agents secreted by the mesenchymal stem cells,can also be suited for use in place of, or as an adjunct to therapy withthe mesenchymal stem cells themselves.

The term “treating” refers to inhibiting, preventing or arresting thedevelopment of a pathology (disease, disorder or condition) and/orcausing the reduction, remission, or regression of a pathology. Those ofskill in the art will understand that various methodologies and assayscan be used to assess the development of a pathology, and similarly,various methodologies and assays may be used to assess the reduction,remission or regression of a pathology.

As used herein, the term “preventing” refers to keeping a disease,disorder or condition from occurring in a subject who may be at risk forthe disease, but has not yet been diagnosed as having the disease.

According to one embodiment, the disease or disorder is selected fromthe group consisting of a bone or cartilage disease, a neurodegenerativedisease, a cardiac disease, a hepatic disease, cancer, nerve damage,wound healing, autoimmune disease, graft versus host disease, spinalcord injury and tissue regeneration.

Bone defects suitable for treatment using the cells of the presentinvention include, but are not limited to osteogenesis imperfecta,fracture, congenital bone defects, and the like.

The conditioned medium of the present invention can be used to treat CNSdiseases. Representative examples of CNS diseases or disorders that canbe beneficially treated with the cells described herein include, but arenot limited to, a pain disorder, a motion disorder, a dissociativedisorder, a mood disorder, an affective disorder, a neurodegenerativedisease or disorder and a convulsive disorder. More specific examples ofsuch conditions include, but are not limited to, Parkinson's, ALS,Multiple Sclerosis, Huntingdon's disease, autoimmune encephalomyelitis,diabetic neuropathy, glaucomatous neuropathy, macular degeneration,action tremors and tardive dyskinesia, panic, anxiety, depression,alcoholism, insomnia, manic behavior, Alzheimer's and epilepsy. Theconditioned medium of the present invention may be suitable for thetreatment of joint conditions including, but not limited toosteoarthritis, rheumatoid arthritis, inflammatory arthritis,chondromalacia, avascular necrosis, traumatic arthritis and the like.

Mesenchymal cell culture conditioned medium can be used to augmentengraftment of transplanted hematopoietic or other stem cells andprevent graft-versus-host disease, for example, in bone marrowtransplantation or cell transplantation for tissue repair as inimplantation for myocardial infarct.

Tissue regeneration: Mesenchymal stem cell conditioned medium of thepresent invention can be used for the promotion of tissue regeneration.Administration of conditioned medium has great promise for benefits inregenerative medicine, autoimmune diseases, inflammatory conditions,acute and chronic ischemic conditions reconstructive surgery, tissueengineering, regenerating new tissues and naturally healing diseased orinjured organs.

Mesenchymal stem cell conditioned medium of the present invention can beused to treat an inflammatory disease or condition in a subject.Inflammatory diseases include, but are not limited to, chronicinflammatory diseases and acute inflammatory diseases.

In some embodiments the inflammatory disease or condition is associatedwith hypersensitivity.

Inflammatory Diseases Associated with Hypersensitivity

Examples of hypersensitivity include, but are not limited to, Type Ihypersensitivity, Type II hypersensitivity, Type III hypersensitivity,Type IV hypersensitivity, immediate hypersensitivity, antibody mediatedhypersensitivity, immune complex mediated hypersensitivity, T lymphocytemediated hypersensitivity and DTH.

Type I or immediate hypersensitivity, such as asthma.

Type II hypersensitivity include, but are not limited to, rheumatoiddiseases, rheumatoid autoimmune diseases, rheumatoid arthritis,spondylitis, ankylosing spondylitis, systemic diseases, systemicautoimmune diseases, systemic lupus erythematosus, sclerosis, systemicsclerosis, glandular diseases, glandular autoimmune diseases, pancreaticautoimmune diseases, diabetes, Type I diabetes, thyroid diseases,autoimmune thyroid diseases, Graves' disease, thyroiditis, spontaneousautoimmune thyroiditis, Hashimoto's thyroiditis, myxedema, idiopathicmyxedema; autoimmune reproductive diseases, ovarian diseases, ovarianautoimmunity, autoimmune anti-sperm infertility, repeated fetal loss,neurodegenerative diseases, neurological diseases, neurologicalautoimmune diseases, multiple sclerosis, Alzheimer's disease, myastheniagravis, motor neuropathies, Guillain-Barre syndrome, neuropathies andautoimmune neuropathies, myasthenic diseases, Lambert-Eaton myasthenicsyndrome, paraneoplastic neurological diseases, cerebellar atrophy,paraneoplastic cerebellar atrophy, non-paraneoplastic stiff mansyndrome, cerebellar atrophies, progressive cerebellar atrophies,encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis,Sydeham chorea, Gilles de la Tourette syndrome, polyendocrinopathies,autoimmune polyendocrinopathies; neuropathies, dysimmune neuropathies;neuromyotonia, acquired neuromyotonia, arthrogryposis multiplexcongenita, cardiovascular diseases, cardiovascular autoimmune diseases,atherosclerosis, myocardial infarction thrombosis, granulomatosis,Wegener's granulomatosis, arteritis, Takayasu's arteritis and Kawasakisyndrome; anti-factor VIII autoimmune disease; vasculitises, necrotizingsmall vessel vasculitises, microscopic polyangiitis, Churg and Strausssyndrome, glomerulonephritis, pauci-immune focal necrotizingglomerulonephritis, crescentic glomerulonephritis; antiphospholipidsyndrome; heart failure, agonist-like β-adrenoceptor antibodies in heartfailure, thrombocytopenic purpura; hemolytic anemia, autoimmunehemolytic anemia, gastrointestinal diseases, autoimmune diseases of thegastrointestinal tract, intestinal diseases, chronic inflammatoryintestinal disease, celiac disease (, autoimmune diseases of themusculature, myositis, autoimmune myositis, Sjogren's syndrome; smoothmuscle autoimmune disease, hepatic diseases, hepatic autoimmunediseases, autoimmune hepatitis and primary biliary cirrhosis.

Type IV or T cell mediated hypersensitivity, include, but are notlimited to, rheumatoid diseases, rheumatoid arthritis, systemicdiseases, systemic autoimmune diseases, systemic lupus erythematosus,glandular diseases, glandular autoimmune diseases, pancreatic diseases,pancreatic autoimmune diseases, Type 1 diabetes; thyroid diseases,autoimmune thyroid diseases, Graves' disease; ovarian diseases,prostatitis, autoimmune prostatitis, polyglandular syndrome, autoimmunepolyglandular syndrome, Type I autoimmune polyglandular syndrome,neurological diseases, autoimmune neurological diseases, multiplesclerosis, neuritis, optic neuritis, myasthenia gravis, stiff-mansyndrome, cardiovascular diseases, cardiac autoimmunity in Chagas'disease, autoimmune thrombocytopenic purpura, anti-helper T lymphocyteautoimmunity, hemolytic anemia, hepatic diseases, hepatic autoimmunediseases, hepatitis, chronic active hepatitis, biliary cirrhosis,primary biliary cirrhosis, nephric diseases, nephric autoimmunediseases, nephritis, interstitial nephritis, connective tissue diseases,ear diseases, autoimmune connective tissue diseases, autoimmune eardisease , disease of the inner ear, skin diseases, cutaneous diseases,dermal diseases, bullous skin diseases, pemphigus vulgaris, bullouspemphigoid and pemphigus foliaceus.

Examples of delayed type hypersensitivity include, but are not limitedto, contact dermatitis and drug eruption.

Examples of types of T lymphocyte mediating hypersensitivity include,but are not limited to, helper T lymphocytes and cytotoxic Tlymphocytes.

Examples of helper T lymphocyte-mediated hypersensitivity include, butare not limited to, T_(h)1 lymphocyte mediated hypersensitivity andT_(h)2 lymphocyte mediated hypersensitivity.

The conditioned medium of the present invention may be used for treatingautoimmune diseases.

Autoimmune diseases include, but are not limited to, autoimmunecardiovascular diseases, rheumatoid diseases, autoimmune glandulardiseases, autoimmune gastrointestinal diseases, autoimmune cutaneousdiseases, autoimmune hepatic diseases, autoimmune neurological diseases,autoimmune muscular diseases, autoimmune nephric diseases, autoimmunediseases related to reproduction, autoimmune connective tissue diseasesand autoimmune systemic diseases.

Examples of autoimmune cardiovascular diseases include, but are notlimited to atherosclerosis, myocardial infarction, thrombosis, Wegener'sgranulomatosis, Takayasu's arteritis, Kawasaki syndrome , anti-factorVIII autoimmune disease, necrotizing small vessel vasculitis,microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focalnecrotizing and crescentic glomerulonephritis, antiphospholipidsyndrome, antibody-induced heart failure, thrombocytopenic purpura,autoimmune hemolytic anemia, cardiac autoimmunity in Chagas' disease andanti-helper T lymphocyte autoimmunity.

Examples of autoimmune rheumatoid diseases include, but are not limitedto rheumatoid arthritis and ankylosing spondylitis.

Examples of autoimmune glandular diseases include, but are not limitedto, pancreatic disease, Type I diabetes, thyroid disease, Graves'disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto'sthyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmuneanti-sperm infertility, autoimmune prostatitis and Type I autoimmunepolyglandular syndrome. diseases include, but are not limited toautoimmune diseases of the pancreas, Type 1 diabetes, autoimmune thyroiddiseases, Graves' disease, spontaneous autoimmune thyroiditis,Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity,autoimmune anti-sperm infertility, autoimmune prostatitis and Type Iautoimmune polyglandular syndrome.

Examples of autoimmune gastrointestinal diseases include, but are notlimited to, chronic inflammatory intestinal diseases, celiac disease,colitis, ileitis and Crohn's disease.

Examples of autoimmune cutaneous diseases include, but are not limitedto, autoimmune bullous skin diseases, such as, but are not limited to,pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.

Examples of autoimmune hepatic diseases include, but are not limited to,hepatitis, autoimmune chronic active hepatitis, primary biliarycirrhosis (and autoimmune hepatitis.

Examples of autoimmune neurological diseases include, but are notlimited to, multiple sclerosis, Alzheimer's disease, myasthenia gravis,neuropathies, motor neuropathies; Guillain-Barre syndrome and autoimmuneneuropathies, myasthenia, Lambert-Eaton myasthenic syndrome;paraneoplastic neurological diseases, cerebellar atrophy, paraneoplasticcerebellar atrophy and stiff-man syndrome; non-paraneoplastic stiff mansyndrome, progressive cerebellar atrophies, encephalitis, Rasmussen'sencephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles dela Tourette syndrome and autoimmune polyendocrinopathies; dysimmuneneuropathies; acquired neuromyotonia, arthrogryposis multiplexcongenita, neuritis, optic neuritis and neurodegenerative diseases.

Examples of autoimmune muscular diseases include, but are not limitedto, myositis, autoimmune myositis and primary Sjogren's syndrome andsmooth muscle autoimmune disease.

Examples of autoimmune nephric diseases include, but are not limited to,nephritis and autoimmune interstitial nephritis.

Examples of autoimmune diseases related to reproduction include, but arenot limited to, repeated fetal loss.

Examples of autoimmune connective tissue diseases include, but are notlimited to, ear diseases, autoimmune ear diseases and autoimmunediseases of the inner ear.

Examples of autoimmune systemic diseases include, but are not limitedto, systemic lupus erythematosus and systemic sclerosis.

The conditioned medium can be used for treating infectious diseases.

Examples of infectious diseases include, but are not limited to, chronicinfectious diseases, subacute infectious diseases, acute infectiousdiseases, viral diseases, bacterial diseases, protozoan diseases,parasitic diseases, fungal diseases, mycoplasma diseases and priondiseases.

The conditioned medium can be used for treating graft rejection.Examples of diseases associated with transplantation of a graft include,but are not limited to, graft rejection, chronic graft rejection,subacute graft rejection, hyperacute graft rejection, acute graftrejection and graft versus host disease.

The conditioned medium can be used for treating allergic conditions ordiseases. Examples of allergic diseases include, but are not limited to,asthma, hives, urticaria, pollen allergy, dust mite allergy, venomallergy, cosmetics allergy, latex allergy, chemical allergy, drugallergy, insect bite allergy, animal dander allergy, stinging plantallergy, poison ivy allergy and food allergy.

The conditioned medium can be used for treating cancerous diseases.Examples of cancer include but are not limited to carcinoma, lymphoma,blastoma, sarcoma, and leukemia. Particular examples of cancerousdiseases but are not limited to: Myeloid leukemia such as Chronicmyelogenous leukemia. Acute myelogenous leukemia with maturation. Acutepromyelocytic leukemia, Acute nonlymphocytic leukemia with increasedbasophils, Acute monocytic leukemia. Acute myelomonocytic leukemia witheosinophilia; Malignant lymphoma, such as Birkitt's Non-Hodgkin's;Lymphoctyic leukemia, such as Acute lumphoblastic leukemia. Chroniclymphocytic leukemia; Myeloproliferative diseases, such as Solid tumorsBenign Meningioma, Mixed tumors of salivary gland, Colonic adenomas;Adenocarcinomas, such as Small cell lung cancer, Kidney, Uterus,Prostate, Bladder, Ovary, Colon, Sarcomas, Liposarcoma, myxoid, Synovialsarcoma, Rhabdomyosarcoma (alveolar), Extraskeletel myxoidchonodrosarcoma, Ewing's tumor; other include Testicular and ovariandysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma, Malignantmelanoma, Mesothelioma, breast, skin, prostate, and ovarian.

Filtering and Concentrating Conditioned Media: The conditioned media canbe pre-filtered to remove large particulates, such as cell debris, forexample, using a liquid filter bag with a 2.5 micron rating to produce“filtered media”. For certain applications the filtered media can beconcentrated, for example, by ultrafiltration (suitable cross flowhollow fiber ultrafiltration cartridges are available from A/GTechnology Corp., Needham, Mass.).

Conditioned media, concentrated conditioned medium, extracts andfractions thereof can be used by formulators for preparing compositionscomprising cosmetic, cosmeceutical, or pharmaceutical formulations withcosmetically-acceptable, cosmeceutically-acceptable orpharmaceutically-acceptable carriers. The skilled artisan willappreciate that cosmetically-acceptable carriers,cosmeceutically-acceptable carriers and pharmaceutically-acceptablecarriers may be the same or different, depending on the intendedapplication of the composition.

As used herein, the term “cosmeceutical” refers to a formulation orcomposition comprising at least one biologically active ingredient thathas an effect on the user of the product and at least onecosmeceutically-acceptable carrier. Cosmeceuticals may be viewed ascosmetics that, in certain applications and under appropriateconditions, may provide medicinal or drug-like benefits. In certainapplications, for example, cosmeceuticals may affect the underlyingstructure of the skin, decrease wrinkle depth, or reverse or amelioratethe effect of photooxidation or aging on the skin. Cosmeceuticals may beparticularly useful as skin care products, hair care products, and suncare products. In certain embodiments, cosmeceutical compositionscomprise delivery systems including at least one of liposomes,cyclodextrins, polymer systems, or hyaluronic acid or related compounds.Cosmeceutical compositions comprise cosmeceutically-acceptable carriers.The skilled artisan will understand that a pharmaceutically-acceptablecarrier or formulation that is suitable for topical applications willtypically also be a cosmeceutically-acceptable carrier or formulation.

A topical cosmetic or cosmeceutical ointment, lotion, or gel compositiontypically contains an effective amount of conditioned media or extractsthereof and may comprise other active and inert ingredients as well in acosmetically- or a cosmeceutically-acceptable carrier, such as apharmaceutical cream base, an oil-in-water emulsion, a water-in-oilemulsion, a gel, or the like. Various cosmetic and cosmeceuticalcompositions for topical use include drops, tinctures, lotions, creams,salves, serums, solutions, and ointments containing conditioned media orextracts, and an appropriate carrier. The optimal percentage of theconditioned media or extract in each composition varies according to thecomposition's formulation and the therapeutic effect desired.

The skilled artisan in the formulation arts will understand that theinventive compositions may comprise any of a number of cosmetically-,cosmeceutically-, or pharmaceutically-acceptable formulations, dependingon the type of product, the nature of the composition, the location ofcomposition's use, the desired effect, and the like.

While proprietary formulations are common in the formulation arts,formulators of ordinary skill will be able to determine or readilyselect appropriate formulations for specific applications without undueexperimentation.

Detailed description of cosmetic- and cosmeceutically-acceptableingredients and formulations may be found in, among other places, FDACosmetics Handbook, U.S. Food and Drug Administration; Handbook ofCosmetic and Personal Care Additives, Ash and Ash, compilers, 1994,Chemical Publishing, New York, N.Y.; Bennett's Cosmetic Formulary, 1993,Chemical Publishing Co.; Harry's Cosmeticology, 7th ed., Wilkinson &Moore, 1982 and 8th ed., Rieger, 2000, Chemical Publishing; CosmeticBench Reference-2001, Allerud Publishing Corp.; CTFA Compendium ofCosmetic Ingredient Composition, Nikitakis and McEwen, eds., 1990,Cosmetic, Toiletry, and Fragrance Association, Washington, D.C.,Surfactant Encyclopedia, 2nd revised edition, Rieger, 1996, AlluredPublishing; The Chemistry and Manufacture of Cosmetics, 2nd ed., DeNavarre, Van Nostrand, Princeton, N.J.; Encyclopedia of Common NaturalIngredients Used in Food, Drugs, and Cosmetics, Leung, 1996, John Wiley;A Consumer's Dictionary of Cosmetic Ingredients, 5th ed., Winter, 1999,Three Rivers Press, New York, N.Y.; Cosmeceuticals: Active SkinTreatment, 1998, Allured Publishing; Handbook of Cosmetic Science andTechnology, Knowlton and Pearce, 1993, Elsevier Advanced Technology,Oxford, UK; Personal-Care Formulas, 1997, Allured Publishing; BeginningCosmetic Chemistry, Scheuller and Romanowski, 1999, Allured Publishing;and Skin Permeation: Fundamentals and Application, Zatz, 1993, AlluredPublishing. Discussions of pharmaceutically-acceptable ingredients andformulations may be found in, among other places, Remington'sPharmaceutical Sciences, 18th ed., Gennaro, ed., 1990, Mack Publishing.

The compositions and methods of the invention have wide applicability tocosmetic conditions. The mode of administration for cosmeticapplications is typically topical, but administration and dosageregimens will vary depending on the cosmetic condition whose modulationis sought. The present invention provides methods, compositions, andkits for cosmetic use with individuals. The term “individual” as usedherein includes humans as well as other mammals. In some embodiments,the compositions, methods, and/or kits are used to provide a cosmetictreatment to an individual desiring and/or in need of cosmetic treatment(e.g., young children subject to burn or other scarring may not desiretreatment but may nonetheless be in need of treatment). The term“treating” or “treatment” as used herein includes achieving a cosmeticbenefit. By cosmetic benefit is meant any desired modulation of thecosmetic condition being treated. For example, in an individual withwrinkling, cosmetic benefit includes eradication or lessening of theappearance of wrinkling. Also, a cosmetic benefit is achieved with theeradication or amelioration of one or more of the psychological symptomsassociated with the underlying condition such that an improvement isobserved in the patient, notwithstanding the fact that the patient maystill be affected by the cosmetic condition. For example, conditionedmedium provides cosmetic benefit not only when a cosmetic defect iseradicated, but also when an improvement is observed in the individualwith respect to the cosmetic defect and its attendant consequences, suchas psychological consequences. In some cases, methods and compositionsof the invention may be directed at achieving a prophylactic benefit. A“prophylactic,” or “preventive” effect includes prevention of acondition, retarding the progress of a condition (e.g., skin aging), ordecreasing the likelihood of occurrence of a condition. As used herein,“treating” or “treatment” includes prophylaxis.

The conditioned medium of the invention, active fractions or extractsthereof can be formulated in a pharmaceutically acceptable carrier,excipient or diluent. The use of such carriers and diluents is wellknown in the art.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA-approved kit, which may containone or more unit dosage forms containing the active ingredient (e.g.,cells). The pack may, for example, comprise metal or plastic foil, suchas a blister pack. The pack or dispenser device may be accompanied byinstructions for administration. The pack or dispenser device may alsobe accompanied by a notice in a form prescribed by a governmental agencyregulating the manufacture, use, or sale of pharmaceuticals, whichnotice is reflective of approval by the agency of the form of thecompositions for human or veterinary administration. Such notice, forexample, may include labeling approved by the U.S. Food and DrugAdministration for prescription drugs or of an approved product insert.Compositions comprising a preparation of the invention formulated in apharmaceutically acceptable carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition, as further detailed above.

The conditioned medium of the present invention or prepared according tothe methods of the present invention, or extracts, fractions or portionsthereof can be administered to the subject per se, and/or in apharmaceutical composition where it is mixed with suitable carriers orexcipients.

As used herein, a “pharmaceutical composition” refers to a preparationof one or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier,” which may be usedinterchangeably, refer to a carrier or a diluent that does not causesignificant irritation to an organism and does not abrogate thebiological activity and properties of the administered compound. Anadjuvant is included under these phrases.

Herein, the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils, and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found inthe latest edition of “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., which is herein fully incorporated byreference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal, or parenteraldelivery, including intramuscular, subcutaneous, and intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations that can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

Pharmaceutical compositions suitable for use in the context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose.More specifically, a “therapeutically effective amount” means an amountof active ingredients (e.g., a nucleic acid construct) effective toprevent, alleviate, or ameliorate symptoms of a disorder (e.g.,ischemia) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, the dosage orthe therapeutically effective amount can be estimated initially from invitro and cell culture assays. For example, a dose can be formulated inanimal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration, and dosage canbe chosen by the individual physician in view of the patient's condition(See, e.g., Fingl, E. et al. (1975), “The Pharmacological Basis ofTherapeutics,” Ch. 1, p.1.).

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks, oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc. As used herein the term “method” refers to manners, means,techniques and procedures for accomplishing a given task including, butnot limited to, those manners, means, techniques and procedures eitherknown to, or readily developed from known manners, means, techniques andprocedures by practitioners of the chemical, pharmacological,biological, biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Maryland (1989); Perbal, “A Practical Guideto Molecular Cloning”, John Wiley & Sons, New York (1988); Watson etal., “Recombinant DNA”, Scientific American Books, New York; Birren etal. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

EXAMPLES Methods and Experimental Procedures

Isolation: Bone marrow derived and adipose tissue derived mesenchymalcells were isolated based on their plastic adherence potential inexpansion medium containing: High glucose DMEM and 10% Fetal BovineSerum (FBS, Hyclone, Logan, Utah, USA) supplemented with 0.05 mg/mlGentamicin (Sigma) and 2 mM L-glutamine (Biological Industries, Israel).Cells were allowed to adhere for 3-4 days and non-adherent cells werewashed out with medium changes. The medium was further exchanged withfresh medium every 3-4 days.

Maintenance and expansion: Once adherent cells reached approximately80-90% confluency, they were detached with 0.25% trypsin-EDTA (Sigma),washed twice in DMEM and 10% Fetal Bovine Serum, with centrifugation,400 g, 5 minutes, and re-plated at a 1:2 to 1:1000 dilution under thesame culture conditions.

Measurement of cell size: Cell size was measured using Cedex AutomatedCell Counter (Innovatis). The cells were diluted 1:2 in Trypan Blue(Sigma) and cell size was measured automatically under microscope.

Measurement of granularicity: Following trypsin treatment, the cellswere analyzed for granularicity by side scatter FACS.

Measurement of number of cells in culture: Cell number was measuredusing Cedex Automated Cell Counter (Innovatis). The cells were diluted1:2 in Trypan Blue (Sigma) and cell number was measured automaticallyunder microscope.

Surface antigen analysis: At different time points the cells weredetached with 0.25% trypsin-EDTA. The cells were washed with a PBSsolution containing 1% BSA, and stained (at 4° C. for 30 min) witheither fluorescein isothiocyanate (FITC) or phycoerythrin(PE)-conjugated antibodies: 105 PE, 105 FITC (Serotec), 45 FITC, 14FITC, HLA-DR FITC, 106 PE, 31 PE (BD), 34 PE (Dako), 73 PE, HLA class 1PE, 49b PE (Phamingen), 29 PE, 44 PE, 54 FITC, 59 PE, 90 PE (BioLegend).The cells were then washed in the above buffer and analyzed using aFACScalibur® flow cytometer (Becton Dickinson). The cells were passed ata rate of up to 1000 cells/second, using a 488 nm argon laser beam asthe light source for excitation. Emission of 10000 cells was measuredusing logarithmic amplification, and analyzed using the CellQuestsoftware (Becton Dickinson). Cells stained with FITC- and PE-conjugatedisotype control antibodies were used to determine backgroundfluorescence.

CFU-F assay: Cultured MSCs were seeded in 6-well plates at density of50-100 cells/cm² and maintained with DMEM and 10% FBS. After 14 days thecells were fixed using 10% cold Formalin (Sigma) and stained with HarrisHematoxylin (Sigma). Clones (cluster of more than 50 cells with evidentepicenter) are stained blue-purple and counted using microscope.

Senescence evaluation assay: Cultured MSCs were stained using theSenescence beta-Galactosidase Staining Kit (Cell Signaling). The cellsare fixed and stained for detection of beta-Galactosidase activity at pH6 using X-Gal and incubation overnight in 37° C. in dry incubator.

In-vitro wound healing assay: Wound was performed in MSCs cultures at˜70% confluence using 200 μl or 1000 μl tip. Four days later the cellswere fixated using 10% cold Formalin (Sigma) and stained with HarrisHematoxylin (Sigma). The in-vitro wound healing process was evaluatedusing microscope.

Treatment of mesenchymal cultures with nicotinamide: Mesenchymalcultures were initiated as described above, and supplemented withnicotinamide 1-15 mM alone, or in combination with growth factors orgrowth factors alone, incubated at 37° C. in a humidified atmosphere of5% CO₂ in air. At each passage and at each medium exchange, the cultureswere supplemented with mesenchymal medium, nicotinamide and growthfactors.

In Vitro Migration Assay: RPMI plus 10% FCS (0.6 ml) containing 100ng/ml CXCL12 (R&D Systems) was placed into the lower chamber of a Costar24-well “transwell” culture plate (Corning, Inc, Corning, N.Y.). Cells(2×10⁵) in 100-μl medium were introduced into the upper chamber, over aporous membrane (pore size, 5 μm). After 4 hours, cells were collectedfrom both chambers and counted by flow cytometry (FACSsort, BectonDickinson and Co, San Jose, Calif., USA). Spontaneous migration wasperformed as a control without CXCL12 in the lower chamber.

In vivo analysis of homing: NOD/SCID mice (8-10 week old) (Harlan Ltd.,Israel) were sub-lethally irradiated (at 375cGy at 67cGy/min) and 24hours later inoculated via the tail vein with either CFSE-labeledmesenchymal stem cells cultured in the presence of nicotinamide orCFSE-labeled mesenchymal stem cells cultured in the absence ofnicotinamide. Mice were sacrificed at 24 hours post injection and bonemarrow or other tissue samples were obtained. Homing of human cells wasdetected by flow cytometry via visualization of CFSE-stained cells overa background of unlabeled murine cells. The bright fluorescence of CFSEis sufficient to separate labeled human cells from unlabeled murinecells by at least 1 log. To quantify homing of human progenitor cells,bone marrow cells were stained with APC-conjugated antihuman cell markermonoclonal antibodies and CFSE⁺/cell marker cells were enumerated. Foreach sample 100,000 events are recorded and analyzed.

Transplantation of mesenchymal cells into NOD/SCID mice: NOD/SCID micewere bred and maintained in sterile intra-ventilated cages (Techniplast,Bugugiatte, Italy). Eight-week-old mice were sub-lethally irradiated asdescribed above. Mice were inoculated via the tail vein with mesenchymalcells cultured in the presence or absence of nicotinamide. To avoiddonor variability, mesenchymal cells from several units were pooled andused for expansion cultures as well as group injection. Mice weresacrificed at week 4, and marrow samples were obtained by flushing theirfemurs and tibias with IMDM at 4° C. Flow cytometric analysis ofNOD/SCID marrow cells was performed as described hereinabove, usingmonoclonal antibodies against human cell surface differentiationantigens to identify human cell engraftment.

Statistics—The non-parametric Wilcoxon Rank Test was applied for testingdifferences between the study groups. All the tests applied weretwo-tailed, and a p value of ≦5% was considered statisticallysignificant. The data were analyzed using SAS software (SAS Institute,Cary, N.C.).

Example 1 Analyzing of Nicotinamide on Mesenchymal Stem Cells Culturedin the Presence of Growth Factors

Materials and Methods

Mesenchymal stem cells were selected and cultured in the presence ofparticular growth factors (basic fibroblast growth factor—bFGF, heparinbinding epidermal growth factor—HB-EGF, fibroblast growth factor 4—FGF-4and platelet derived growth factor, homodimer, subunit B, PDGF-BB) inthe presence and absence of nicotinamide for three or four passages andthe number and size of the cells was calculated.

Two concentrations (10 and 50 ng/ml) of each one of the followingfactors were examined.

The experimental groups were as follows:

Group 1: Ctrl

Group 2: 10 ng/ml growth factor

Group 3: 50 ng/ml growth factor

Group 4: 5 mM NAM

Group 5: 5 mM NAM+10 ng/ml growth factor

Group 6: 5 mM NAM+50 ng/ml growth factor

In addition, the influence of the combination: 5 mM NAM+50 ng/ml FGF4+50ng/ml PDGF-BB was examined in comparison to an individual supplement.

Results

FIG. 1 illustrates that basic FGF has a negative effect on the abilityof nicotinamide to increase proliferation of mesenchymal stem cells.

FIGS. 2A-B illustrate that heparin-binding EGF-like growth factor(HB-EGF) has a negative effect on the ability of nicotinamide toincrease proliferation of mesenchymal stem cells.

FIGS. 3A-3E, 4A-4B and-5A-5D illustrate that nicotinamide has apotentiating effect on the ability of FGF4 to increase proliferation ofmesenchymal stem cells of diverse origins.

FIGS. 6A-D illustrate that PDGF-BB has an inconsistent effect on theability of nicotinamide to increase proliferation of mesenchymal stemcells.

FIGS. 7A-D illustrate that MSC cultures treated with PDGF-BB or acombination of PDGF-BB+NAM are contaminated with a higher fraction ofcells other than MSCs as compared with cultures treated without PDGF-BB.

FIGS. 8A-B, 9A-B and 10A-H illustrate that the combination of threefactors—FGF4, nicotinamide and PDGF-BB has a detrimental effect onproliferation of mesenchymal stem cells as compared to the effect ofFGF4 and nicotinamide in the absence of PDGF-BB.

Example 2 The Effect of Nicotinamide on Bone Marrow Derived MesenchymalStem Cell Culture

The present inventors showed that nicotinamide increased the seedingefficacy (selection) of bone marrow derived MSCs. Phenotpyiccharacterization of these cells after one passage in nicotinamide isshown in FIGS. 11 and 13. FIGS. 3E, 15, 21 illustrate the effect ofnicotinamide on the expansion rate of bone marrow derived MSCs. Lowconcentrations of nicotinamide (e.g. 0.1 mM) had insignificant effect onthe expansion rate of bone marrow derived MSCs (Figure not shown). FIGS.18A-C and 19A-B illustrate that mesenchymal stem cells grown in thepresence of nicotinamide are smaller and less granular than mesenchymalstem cells grown in the absence of nicotinamide under identicalconditions.

Example 3 Nicotinamide Increases Expansion of Cultured AdiposeTissue-Derived Mesenchymal Cells

Phenotypic characterization of adipose tissue derived MSCs is shown inFIG. 12A-12C. As illustrated in FIGS. 14-17, nicotinamide substantiallyimproved adipose derived mesenchymal stem cell expansion in culture.When adipose derived mesenchymal stem cells were cultured in thepresence of nicotinamide and FGF4 (FIGS. 15-17), a synergic enhancementof proliferation of both total cells in the culture (FIG. 15) andnucleated cells in the culture (FIG. 16) was observed. Measurement ofthe size of the expanded adipose-derived mesenchymal stem cells revealedthat nicotinamide enhanced growth of populations of smaller (lessdifferentiated) cells (FIG. 17), and that culture in a combination ofFGF4 and nicotinamide resulted in increased proliferation of populationsof even smaller cells.

Example 4 Further Analysis on the Effect of Nicotinamide on MesenchymalStem Cells

Materials and Methods

Mesenchymal stem cells were isolated using plastic adherence method, asdescribed above and cultured for several passages with fetal bovineserum, ±NAM. The cells were selected in the presence of NAM.

At about 80% confluence, adherent cells were collected following trypsintreatment, counted, characterized and re-seeded at a concentration of1×10³ cells/cm².

Measurement of VCAM1/CD106: Following Trypsin treatment the cells wereanalyzed for CD106 expression in FACS using anti-human CD106 PEantibodies.

Measurement of CD54: Following Trypsin treatment the cells were analyzedfor CD54 expression in FACS using anti-human CD54 antibodies.

Results

Large batches of mesenchymal stem cells cultured with nicotinamide alsoshowed enhanced proliferative capacity, indicating that large commercialbatches of MSCs can be manufactured with fewer passages, thus ensuringshorter cultivation time and higher quality of the therapeutic cells dueto preservation of stem cells characteristics by nicotinamide. Yetfurther, number of senescent cells was reduced following culture innicotinamide. This effect of nicotinamide was not dependent on anyparticular batch of serum (data are not shown).

FIG. 20 illustrates that mesenchymal stem cells grown in the presence ofnicotinamide have higher wound closure capacity than mesenchymal stemcells grown in the absence of nicotinamide under identical conditions.

Example 5 Effect of Combined Nicotinamide and FGF4 on Adipose-derivedMesenchymal Stem Cell in Culture

Proliferation and cell size distribution in adipose-derived mesenchymalstem cells cultured with nicotinamide with or without additional FGF4was assessed in up to 4 passages of the cultures.

FIGS. 15-16 illustrate the striking effect of combined nicotinamide andFGF4 on adipose derived adherent cell proliferation, expressed as thenumber of total nucleated cells in the cultures, compared to controls aswell as nicotinamide-treated cultures.

The size of mesenchymal stem cells in culture is often used as anindicator of the degree of differentiation of the MSCs, with theundifferentiated state more prevalent in the smaller size cells. FIG. 17illustrates the increased prevalence of smaller size cells in culturesof adipose derived MSCs exposed to nicotinamide, and the yet greaterprevalence of smaller size cells among adipose derived MSCs exposed tonicotinamide and FGF4.

Thus, these results indicate that a combination of nicotinamide and FGF4synergistically increases the rate of proliferation of adipose derivedmesenchymal cells, while effectively maintaining the cells in anundifferentiated state.

Example 6 Biologically Active Molecules in Conditioned Medium ofMesenchymal Stem Cells Cultured with Nicotinamide and FGF4

Mesenchymal stem cells cultured with nicotinamide and FGF4 have enhancedexpansion potential, and maintain an undifferentiated state, compared tomesenchymal stem cells cultured in unsupplemented medium, or mesenchymalstem cells cultured with added nicotinamide alone. In order to assesswhether mesenchymal stem cells cultured with nicotinamide ornicotinamide and FGF4 modify the culture medium in a unique manner,secretion of biologically active molecules into the culture medium wasmeasured by ELISA, after depletion of the FGF4 from the medium.

As assayed by ELISA, the conditioned medium from mesenchymal stem cellscultured with nicotinamide or nicotinamide and FGF4 exhibited a distinctprofile of biologically active factors. FIGS. 22-25 illustrate thesignificant increase in hepatocyte growth factor (HGF, FIG. 22),transforming growth factor beta (TGF-β, FIG. 23) and keratinocyte growthfactor (KGF, FIG. 24) with combined FGF4 and nicotinamide, compared tonicotinamide alone. FIG. 25 shows nicotinamide's reduction inpro-inflammatory interleukin 6 (IL-6) secreted, and the furtherreduction in IL6 with addition of FGF4.

Thus, mesenchymal stem cells cultured with nicotinamide or nicotinamideand FGF4 produce conditioned medium characterized by increasedconcentration of growth factors and reduced pro-inflammatory factors.

Example 7 Effect of Conditioned Medium of Mesenchymal Stem CellsCultured with Nicotinamide and FGF4 on Inflammatory Processes

Conditioned medium from mesenchymal stem cells cultured withnicotinamide or nicotinamide and FGF4 was assayed for anti-inflammatoryactivity in both in-vivo and in-vitro assays, after depletion of theFGF4 from the medium.

In vivo Delayed Type Hypersensitivity Assay: The in-vivo delayed typehypersensitivity assay measures cutaneous inflammatory response to anallergen challenge and involves the rapid recruitment of macrophages,basophils and T-cells. When medium from mesenchymal stem cell culturedwith nicotinamide was assayed in the in-vivo delayed hypersensitivitytest, reduction in inflammatory response to challenge with thesensitizing allergen (Oxazolone) was clearly observed (see FIGS. 26 and27). Application of conditioned medium from mesenchymal stem cells grownwith FGF4 without nicotinamide resulted in a moderate inhibition of thedelayed type hypersensitivity response. An even greater, synergicinhibition of the delayed-type hypersensitivity reaction was observedwith conditioned medium from mesenchymal stem cell cultured withnicotinamide and FGF4 (FIGS. 28 and 29).

Biological activity of conditioned medium was found to increase withlength on time in contact with the cultured mesenchymal stem cells. Whenconditioned medium from mesenchymal stem cell cultured with nicotinamideand FGF4 was collected 24 or 48 hours following serum depletion andmedium replacement and tested in the delayed type hypersensitivityassay, an increase in immune modulatory capacity of the conditionedmedium (FIG. 30) from 24 to 48 hours in culture was clearly observed,indicating enhanced anti-inflammatory capacity with the extended time inculture. Thus, although conditioned medium clearly has biologicalactivity following 24 hours in culture, optionally, conditioned mediumcollected after greater than 24 hours may have greater or additionallyadvantageous activity.

In vitro Mixed-Lymphocyte-Like Assay: The in-vitro mixed lymphocyte-likeassay measures inflammatory response of peripheral blood mononuclearcells (>50% T-cells) to activation (e.g. by PHA), and allows evaluationof the effect of exogenously added factors on inflammatory processes.When the mononuclear cell medium was supplemented with culture mediumfrom mesenchymal stem cells cultured without nicotinamide or FGF4,secretion of TNFa in response to PHA activation was significantlyelevated, in a dose-dependent manner (see FIG. 32A, columns 3-5).Supplementing with culture medium from mesenchymal stem cells culturedwith nicotinamide and FGF4 clearly inhibited secretion of TNFα in theactivated cells, in a dose-dependent manner (see FIG. 32A, columns 6-8).

When the mononuclear cell medium was repeatedly supplemented withconditioned medium over a 3 day period, even greater elevation ofinflammatory response to PHA activation was observed with conditionedmedium from mesenchymal cells cultured without nicotinamide or FGF4 (seeFIG. 32B, columns 3-5). Repeated supplementation with conditioned mediumfrom mesenchymal cells cultured with nicotinamide and FGF4 (see FIG.32B, columns 6-8) strongly inhibited the mononuclear cell's inflammatoryresponse, again in a dose dependent manner. Thus, while conditionedmedium from mesenchymal stem cell culture without nicotinamide and FGF4does not inherently comprise anti-inflammatory potential (at least asmeasured by the mixed lymphocyte reaction), conditioned medium frommesenchymal stem cell culture from MSC cultured with nicotinamide andFGF4 has distinct, quantifiable biological properties (e.g.anti-inflammatory) which result from the culturing with nicotinamide andFGF4.

Example 8 Conditioned Medium of Mesenchymal Stem Cells Cultured withNicotinamide and FGF4 as Growth Supplement

Culture of mesenchymal stem cells with nicotinamide or nicotinamide andFGF4 resulted in increased secretion of some growth factors into themedium (see Example 6 above). Conditioned medium from mesenchymal stemcell cultured with nicotinamide or nicotinamide and FGF4 was assayed forits effect on keratinocyte proliferation in-vitro.

FIG. 31A shows the striking enhancement of normal human epidermalkeratinocyte proliferation in-vitro with addition of 10% v/v conditionedmedium from mesenchymal stem cell cultured with 5 mM nicotinamide,significantly greater than the effect of conditioned medium frommesenchymal stem cells cultured without nicotinamide. From the enhancedkeratinocyte proliferation with +NAM+FGF4 conditioned medium (see FIG.31B) it is clear that mesenchymal stem cells grown with bothnicotinamide and FGF4 to the mesenchymal stem cell produce a culturedmedium having even greater mitogenic potential (see FIG. 31B) than thatof conditioned medium from mesenchymal cells cultured with nicotinamidealone.

These results indicate that culture medium conditioned by adherentmesenchymal stem cells cultured with nicotinamide or nicotinamide incombination with FGF4 contains biologically active factors, includingfactors having anti-inflammatory and proliferation-inducing (mitogenic)activity.

Example 9 Conditioned Medium from Nicotinamide-Treated Mesenchymal StemCell Cultures Increases Functionality of Chemokine Receptors andAdhesion Molecules

In order to determine the role of adhesion and related molecules in theeffects of conditioned medium from nicotinamide- andnicotinamide-FGF4-treated mesenchymal stem cell cultures on homing andengraftment of cells, the effect of conditioned medium on in-vitromigration and the functionality of the adhesion molecules can be tested.

Using a trans-well migration assay, CXCL12-induced migration of cellsexposed to conditioned medium (e.g mesenchymal stem cells) is tested,assessing the effects of conditioned medium on integrin and adhesionmolecule function. Enhanced stimulation of migration by the conditionedmedium from nicotinamide and/or nicotinamide-FGF4 treated cells,compared to conditioned medium from cells cultured without nicotinamideor non-cultured cells suggests that treatment of mesenchymal cells withthe conditioned medium from nicotinamide-treated cells can potentiallyincrease the responsiveness of adhesion molecules to their ligands,resulting in enhanced engraftment and homing potential of theconditioned-medium-treated cells.

The functional quality of cell binding to adhesion molecules can also beinvestigated using shear flow analysis. The strong effect of conditionedmedium of nicotinamide- and/or nicotinamide and FGF4-treated cells onadhesion molecule-mediated binding and retention on substrate adhesionmolecules can be revealed by a significantly enhanced percentage ofinitially settled cells resistant to removal by shear stress evident inthe cells exposed to conditioned medium from mesenchymal cells treatedwith nicotinamide or nicotinamide and FGF4.

Example 10 Cultured Medium from Nicotinamide-Treated Mesenchymal StemCell Cultures Increases the SCID-Repopulating Capacity of Cultured Cells

Conditioned medium from nicotinamide- and nicotinamide-FGF4-treatedmesenchymal stem cell cultures is tested for ability to enhance homingand engraftment of transplanted cells by repopulation of NOD/SCID mice.To evaluate repopulating capacity, NOD/SCID mice are transplanted withmesenchymal cells exposed, for different periods of time to conditionedmedium from nicotinamide- and nicotinamide-FGF4-treated mesenchymal stemcell cultures, as well as untreated, non-cultured mesenchymal cells andmesenchymal cells cultured in sham conditioned medium (conditionedmedium from cells cultured in cytokines only). The cells aretransplanted over a range of doses intended to achieve a sub-optimaltransplantation, and subsequent non-engraftment in a fraction of mice.Human cell engraftment is evaluated 4-weeks post transplantation. Miceare scored as positively engrafted if 0.5% of the recipient bone marrowcells expressed human CD45 antigen (CD45+). In the event that exposureto conditioned medium from nicotinamide- and nicotinamide-FGF4-treatedmesenchymal stem cell cultures results in superior and clear engraftmentof mesenchymal cells in the mice at a predetermined dose range, whilethe untreated cells fail to engraft or engraft poorly, it can beconcluded that the conditioned medium is effective in enhancingengraftment and homing of transplanted mesenchymal cells.

Example 11 Nicotinamide Increases Tissue Homing of Cultured MesenchymalCells

To evaluate the effect of conditioned medium from nicotinamide- andnicotinamide-FGF4-treated mesenchymal stem cell cultures on the homingof cultured mesenchymal cells, NOD/SCID mice are transplanted withmesenchymal cells exposed, for different periods of time to conditionedmedium from nicotinamide- and nicotinamide-FGF4-treated mesenchymal stemcell cultures, as well as untreated, non-cultured mesenchymal cells andmesenchymal cells cultured in sham conditioned medium (conditionedmedium from cells cultured in cytokines only). Prior to transplantation,the cells are labeled with CFSE. Twenty-four hours post transplantationtotal CFSE-labeled cells and CFSE labeled mesenchymal cells that homedto the mouse bone marrow of the recipient mice are quantified by FACS.

Results indicate an effect of the conditioned medium on tissue homing ofmesenchymal cells, if the homing of the conditioned medium-treatedmesenchymal cells is significantly higher than the homing of mesenchymalcells not exposed to conditioned medium.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications and GenBank Accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application or GenBank Accession numberwas specifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1. A method of preparing a conditioned cell culture medium, the methodcomprising (a) culturing a population of the mesenchymal stem cells in amedium comprising nicotinamide, and, optionally, fibroblast growthfactor 4 (FGF4); and (b) collecting the conditioned cell culture medium.2-3. (canceled)
 4. The method of claim 1, wherein said culturing iseffected in a medium comprising DMEM.
 5. (canceled)
 6. The method ofclaim 1, wherein the mesenchymal stem cells are derived from a tissueselected from the group consisting of bone marrow, adipose tissue,placenta and umbilical cord blood. 7-8. (canceled)
 9. The method ofclaim 8, wherein said mesenchymal stem cells are plastic-adherent cells.10-11. (canceled)
 12. The method of claim 1, wherein a calciumconcentration of said medium is greater than 1.8 mM. 13-14. (canceled)15. The method of claim 1, wherein a concentration of said nicotinamideis 1-20 mM.
 16. The method of claim 1, wherein a concentration of saidFGF4 is 10-100 ng/ml.
 17. The method of claim 1, wherein said culturingis effected in a medium being devoid of platelet derived growth factor(PDGF) or fibroblast growth factor 2 (FGF2) or both.
 18. The method ofclaim 1, wherein said culturing said population of mesenchymal stemcells comprises culturing said population of mesenchymal stem cells fora first period of time in a medium devoid of nicotinamide; and thenculturing said population for a second period of time in a mediumcomprising nicotinamide and FGF4.
 19. (canceled)
 20. The method of claim18, wherein said medium devoid of nicotinamide is devoid of FGF4. 21.The method of claim 18, wherein said medium devoid of nicotinamidecomprises FGF4. 22-24. (canceled)
 25. The method of claim 1, whereinsaid mesenchymal stem cells are cultured for a first period of time in aculture medium comprising serum followed by a second period of time in aserum-free culture, and wherein said conditioned medium is collectedfrom the culture of the second period of time.
 26. (canceled)
 27. Amesenchymal stem cell conditioned medium produced by the method ofclaim
 1. 28. (canceled)
 29. The conditioned medium of claim 27, whereinsaid conditioned medium is characterized by an increased level of atleast one biologically active factor selected from the group consistingof HGF, KGF and TGFβ, compared to levels of said at least one factor inconditioned medium from mesenchymal stem cells cultured without addednicotinamide.
 30. A pharmaceutical composition comprising theconditioned medium of claim 27, or a biologically active fractionthereof, and a pharmaceutically acceptable excipient, diluent orcarrier.
 31. A cosmeceutical composition comprising the conditionedmedium of claim 27, or a biologically active fraction thereof and acosmetically acceptable excipient, diluent or carrier.
 32. A method oftreating an inflammatory disease in a subject in need thereof,comprising administering to the subject the conditioned medium of claim27, for use in treating an inflammatory disease in a subject in needthereof.
 33. (canceled)
 34. A method of culturing cells, the methodcomprising culturing the cells in a culture medium comprising theconditioned medium of claim
 27. 35. The method of claim 34, wherein saidcells are keratinocytes.
 36. A cell culture comprising cells and aculture medium, the medium comprising the conditioned medium of claim27.